Biol. Cell (2014) 106, 359–376 DOI: 10.1111/boc.201400013 Research article

Human RAD23 homolog A isrequired for the nucleartranslocation of apoptosis-inducingfactor during induction of cell deathJanaki N. Sudhakar* and Kuan-Chih Chow*†1

*Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China and †Agricultural

Biotechnology Centre, National Chung Hsing University, Taichung, Taiwan, Republic of China

Background Information. During the initiation of cell death, mitochondrial protein, apoptosis-inducing factor (AIF),is transported to the nucleus. The mechanism of AIF nuclear translocation, however, is not clear. After proteinsynthesis, the AIF is originally targetted to the mitochondria, and the nuclear targetting is a secondary event.Therefore, we hypothesised that the nuclear translocation of AIF could be achieved by a novel pathway.

Results. By using yeast two-hybrid assay, we identified the human UV excision repair protein RAD23 homologA (hHR23A) interacts with AIF and their interaction was confirmed by co-immunoprecipitation and fluorescenceresonance energy transfer microscopy. Silencing the RAD23A gene expression inhibits the nuclear transportation ofAIF and increases cisplatin resistance. Silencing the karyopherin alpha 2 (KPNA2) gene expression, however, did notaffect the nuclear import of AIF. Moreover, 2,4-dinitrophenol inhibits staurosporine-induced nuclear translocationof AIF and increases cisplatin resistance.

Conclusions. These results suggest that hHR23A is required for the nuclear translocation of AIF during inductionof cell death, and this process is energy dependent, but independent of karyopherins.

� Additional supporting information may be found in the online version of this article at the publisher’sweb-site

IntroductionApoptosis-inducing factor (AIF), a 67-kDa flavopro-tein, is located in the intermembrane space (IMS) asa nicotinamide adenine dinucleotide-dependent oxi-doreductase of the mitochondrial respiratory chain(Modjtahedi et al., 2006). Because of the presence

1To whom correspondence should be addressed (emailkcchow@dragon.nchu.edu.tw)Key words: Apoptosis-inducing factor, Cisplatin resistance, FRET, hHR23A,Nuclear translocation.Abbreviations: AIF, apoptosis-inducing factor; CIM, confocal immunofluores-cence microscopic; cNLS, classical nuclear localisation signal; CNS, cytoplasm-nucleus shuttle; DNP, 2,4-dinitrophenol; FRET, fluorescence resonance energytransfer; hHR23A, human UV excision repair protein RAD23 homolog A; IMS, in-termembrane space; KPNA2, karyopherin subunit alpha-2; MALDI-TOF, matrix-assisted laser desorption/ionisation time of flight; MEFs, mouse embryonicfibroblasts; Microsomal GST-1, microsomal glutathione S-transferase 1; NPC,nuclear–pore complexes; STS, staurosporine; TCS, ternary complex shuttle;WCL, whole cell lysates.

of two flavin adenine dinucleotide-binding motifswithin the protein structure, AIF was considered toplay a critical role in the biosynthesis of ATP andthe removal of free radicals (Vahsen et al., 2004;Cheung et al., 2006). Following genotoxic stress(e.g. cytotoxic drug challenge, hypoxia and irradia-tion), AIF is released from the mitochondria, andtranslocated to the nucleus to initiate the nuclearevents of apoptosis (Daugas et al., 2000; Susin et al.,2000). Beside the nuclear events, a number of com-monly used anticancer drugs (e.g. epipodophyllotox-ins, camptothecin and cisplatin) were also shown toincrease the permeabilisation of mitochondrial outermembrane, induce protein efflux from the IMS andactivate caspase-related apoptosis (Susin et al., 2000).Depending upon the involvement of caspase, theapoptosis is further divided into caspase-dependent

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Sudhakar and Chow

and -independent cell death (Joseph et al., 2002).Following efflux of the IMS proteins, cytochromec activates the apoptotic protease-activating factor 1and initiates caspase-dependent cell death throughthe subsequent caspase cascade (Brustugun et al.,1998). Although caspase-independent cell death hasbeen suggested to be mediated by the nuclear translo-cation of AIF (Susin et al., 2000; Vahsen et al., 2004),the detailed mechanism of AIF translocation from themitochondria to the nucleus is not well characterised.

In this study, we used the yeast two-hybrid as-say to screen for proteins that interacted with AIFfrom both the cytoplasmic and the nuclear libraries,and we identified the human UV excision repairprotein RAD23 homolog A (hHR23A). We raisedthe monoclonal antibodies specific to AIF (Chenet al., 2006) and hHR23A (Chiang et al., 2009),respectively. Following immunoprecipitation (IP),identities of the co-precipitated proteins were de-termined by using the matrix-assisted laser des-orption/ionisation time of flight mass spectrometry(MALDI-TOF). Silencing the human RAD23 ho-molog A (Saccharomyces cerevisiae) (RAD23A) geneexpression by using small hairpin RNA (shRNA) in-hibited staurosporine (STS)-induced nuclear translo-cation of the AIF, and reduced cisplatin-mediatedcytotoxicity. Specific binding segments between AIFand hHR23A were identified by deletion studies andthe direct binding between the two proteins were fur-ther determined by using confocal fluorescence reso-nance energy transfer (FRET) microscopy. Our resultssuggested that hHR23A is required for the nucleartransport of AIF during initiation of apoptosis.

ResultsAIF interacts with hHR23AUsing AIF as bait in the yeast two-hybrid assay, wescreened libraries containing cytoplasmic (ModulesLPap002, LPap003 and LPap005, Level Biotechno-logy Inc., Taipei, Taiwan) or nuclear (ModuleLPtf010) proteins and we identified three proteinswhich interact with AIF. They are hHR23A, micro-somal glutathione S-transferase 1 (Microsomal GST-1) and caspase-14 (CASP-14) (Fang et al., 2011) inthe cytoplasmic library; and hHR23A in the nuclearlibrary (Table 1).

The cDNA fragments of AIF and RAD23A wereamplified from the lung cancer cDNA library and

Figure 1 Characterisation of the monoclonal antibodiesspecific to hHR23A(A) Whole cell lysates of A549 cells were used to run the

two-dimensional gel electrophoresis and were analysed by

Coomassie Brilliant Blue staining (left panel) and immunoblot-

ting with the monoclonal antibodies specific to hHR23A (right

panel). The white arrows indicate the two protein spots with

molecular masses of 58-kDa (pH 5.0) and 64-kDa (pH 5.7).

(B) Whole cell lysates of A549 cells were incubated with the

monoclonal antibodies specific to AIF or hHR23A and the im-

munocomplexes were precipitated by using resin-conjugated

protein G and resolved by immunoblotting with anti-AIF (the

left panel) or anti-hHR23A (the right panel) antibodies. IP, im-

munoprecipitation; IB, immunoblotting.

then subcloned into pET32a (+) bacterial expres-sion vectors, respectively. The antibodies specificto hHR23A were raised against the recombinanthHR23A proteins and characterised by immunoblot-ting, which recognised two protein spots with molec-ular masses of 58-kDa (pH 5.0) and 64-kDa (pH 5.7)(Figure 1A) in a two-dimensional gel electrophoresisof the A549 cell lysates. The 58-kDa protein was fur-ther analysed by using MALDI-TOF, and their pep-tide mass fingerprints matched that of the hHR23A(UniProtKB/Swiss-Prot, P54725, UV excision re-pair protein RAD23 homolog A). The 64-kDa pro-tein band is human UV excision repair proteinRAD23 homolog B (hHR23B) (UniProtKB/Swiss-Prot, P54727, UV excision repair protein RAD23homolog B). The specificity of the antibodies tohHR23A was further confirmed by immunoblottingthe whole cell lysates of Rad23a−/− or Rad23b−/−

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hHR is required for AIF transport Research article

Table 1 Identification of proteins interacting with AIF.The proteins that interact with AIF were identified from the libraries containing cytoplasmic (Modules LPap002, LPap003 andLPap005, Level Biotechnology Inc., Taipei, Taiwan) and nuclear (Module LPtf010) proteins by using AIF as bait in the yeasttwo-hybrid assay

Growth Selection X-Gal Assay

Module Location Acc. No. Gene Name 1st Repeat 1st Repeat

LPtf005 E8, E9 NM 005053 RAD23 homolog A (S. cerevisiae) (RAD23A) +++ +++ +++ +++LPtf005 F2, F3 NM 020300 microsomal glutathione S-transferase 1 (MGST1) +++ +++ +++ +++LPtf005 F10, F11 NM 012114 caspase 14 (CASP14) +++ +++ +++ +++

LPtf010 E10, E11 NM 005053 RAD23 homolog A (S. cerevisiae) (RAD23A) +++ +++

mouse embryonic fibroblasts (MEFs) cells (Supple-mentary Figure S2 A). The antibodies specific to AIFrecognised a single unique 67-kDa protein band fromthe whole cell lysates of the lung cancer cells (Chenet al., 2008). Characterisation of the monoclonal an-tibodies to AIF was following the routine procedure,and the peptide mass fingerprints matched that of theAIF (UniProtKB/Swiss-Prot, O95831, AIF 1, mito-chondrial) (Chen et al., 2008). Following charac-terisation, the antibodies specific to AIF precipitatedboth AIF and hHR23A, similarly, the antibodies spe-cific to hHR23A co-precipitated hHR23A and AIFfrom the whole cell lysates of A549 cells (Figure 1B).These data indicate that hHR23A interacted withAIF.

Protein expression and subcellular distribution ofhHR23A and AIFNext, we examined the protein expression levels ofhHR23A and AIF in several human cancer cell linesto study the effect of RAD23A gene silencing on AIFactivity. We found hHR23A was highly expressed inseveral lung cancer cell lines, but the levels of AIFvaried among different cell lines (Figure 2A).

The confocal immunofluorescence microscopic(CIM) images and subcellular fractionation of A549control cells showed that AIF was located in the mi-tochondria (Figures 2B and 2C), and hHR23A wasevenly distributed in the cytoplasm and some werelocated in the nuclei (Figures 2C and 2D). Followingtreatment with STS (a pan-protein kinase inhibitorisolated from Streptomyces staurospores, which caspase-independently induces apoptosis) (Zhang et al.,2004), actinomycin D, etoposide and cisplatin inthe presence or absence of broad-spectrum caspaseinhibitor Q-VD-OPH (Caserta et al., 2003; Chau-

vier et al., 2007), the AIF was translocated to thenucleus indicating the nuclear translocation of AIFduring cell death is caspase independent (Figure 2B).Notably, the decrease in the mitochondrial levels ofAIF and increase in the nuclear fraction was muchhigher in STS-treated cells when compared withactinomycin D (Figure 2C), etoposide, and cisplatintreated cells (Figure 2B), indicating STS is more po-tent in inducing the nuclear translocation of AIFduring cell death. Similarly, following STS and acti-nomycin D treatment in the presence or absence ofQ-VD-OPH, the nuclear content of hHR23A in-creased (Figure 2D). Analysis of the subcellular frac-tionation and immunoblotting confirmed our CIMfindings that the nuclear translocation of AIF was as-sociated with the increase in the nuclear content ofhHR23A (Figure 2C). Interestingly, following STSand actinomycin D treatment, the levels of hHR23Aincreased not only in the nuclear fraction but also inthe mitochondrial fraction, indicating that hHR23Acould enter the mitochondria (Figures 2C and 2D).Finally, when compared with the control groups,the nuclear AIF- and hHR23A-positive cells showeddistinctive peripheral chromatin condensation (Fig-ures 2B and 2D) after STS treatment, a feature ofstage I apoptosis where large fragments of degradedDNA conglomerate (Susin et al., 2000).

hHR23A is essential for STS-induced nucleartranslocation of AIF and cisplatin-mediatedcytotoxicityThe hHR23A protein expression was silenced by us-ing RAD23A-gene specific shRNA, which markedlyreduced the 58-kDa hHR23A protein expressionin HeLa and H2087 cells (Figure 3A), but hadno effect on the expression of 64-kDa hHR23B,

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Figure 2 The protein expression levels and subcellular distribution of hHR23A and AIF in the lung cancer cells(A) The protein expression levels of hHR23A and AIF in the seven human lung cancer cell lines were determined by immunoblot-

ting. (B) The confocal immunofluorescence microscopic (CIM) images of A549 control cells show AIF (stained by using AIF

antibody and labelled with Alexa flour 546, red fluorescence) is normally located in the mitochondria (stained by using HSP60

antibody and labelled with Alexa flour 488, green fluorescence). Following STS (1 μM for 3 h), actinomycin D (10 μg/ml for

24 h), etoposide (100 μM for 24 h) and cisplatin (50 μM for 24 h) treatment in the presence or absence of broad-spectrum

caspase inhibitor Q-VD-OPH (50 μM), the amount of nuclear AIF increased (indicated by an increase of pink fluorescence in

the nuclei).The nuclei were counterstained by using 4′, 6-diamidino-2-phenylindole (DAPI). The white arrows indicate peripheral

chromatin condensation after STS treatment. The scale bars represent 10 μm. (C) The subcellular fractionation and immunoblot-

ting analysis show the level of AIF in the mitochondrial fraction decreased while that in the nuclear fraction increased following

STS (1 μM for 3 h) and Act D (Actinomycin D, 10 μg/ml for 24 h) treatment in A549 cells. STS and act D increased the level of

58-kDa hHR23A in both the mitochondrial and the nuclear fractions. (D) The CIM images of A549 control cells show hHR23A

(red fluorescence) is distributed in the cytoplasm and some in the nuclei as well. The arrow head indicates that hHR23A barely

localised in the mitochondria (green fluorescence) of control cells. Following STS (1 μM for 3 h) and Act D (10 μg/ml for 24 h)

treatment in the presence or absence of Q-VD-OPH (50 μM), the nuclear levels of hHR23A increased (indicated by an increase of

pink fluorescence in the nuclei), and the hHR23A and mitochondria too co-localised (arrows indicate an increase of yellow fluo-

rescence in the mitochondria) when compared with the control cells. The white empty arrowheads indicate peripheral chromatin

condensation after STS treatment. The magnified images are of the boxed regions. The scale bars represent 10 μm.

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Figure 2 Continued.

indicating gene specific silencing. Upon STS chal-lenge, the nuclear translocation of AIF was signif-icantly inhibited in RAD23A-silenced HeLa cellswhen compared with the wild-type (WT) or cells in-fected with the mock shRNAs (Figure 3B), and thisfinding was further confirmed by subcellular frac-tionation and immunoblotting of RAD23A-silencedHeLa cells (Supplementary Figure S1). The decreasein the number of AIF-positive nuclei after STS chal-lenge to the RAD23A-silenced HeLa cells was about70–80% when compared with the control cells (Fig-ure 3C). Moreover, cisplatin-mediated cytotoxicitywas also markedly reduced in RAD23A-silencedHeLa cells (Figure 3D), and the IC50 was increasedfrom 1.5 to 2.5 μM cisplatin, suggesting that thepresence of hHR23A was essential for the transporta-tion of AIF into the nucleus to induce apoptosis.

To further confirm this hypothesis, the mouseRAD23 homolog (S. cerevisiae) (Rad23)-knockoutMEFs cell lines were treated with STS. In the MEFWT cells, the STS-induced nuclear translocationof AIF (Figure 3E1) was similar to that found inthe A549 cells (Figure 2B). Interestingly, when themouse RAD23a homolog (S. cerevisiae) (Rad23a) genewas deleted, the nuclear translocation of AIF wasnullified; whereas the deletion of mouse RAD23bhomolog (S. cerevisiae) (Rad23b) gene had no effecton the nuclear translocation of AIF (Figures 3E2 and3E3). The subcellular fractionation and immunoblot-

ting further confirmed that AIF was translocated intothe nucleus upon STS-challenge in MEF WT, andMEF Rad23b−/− cells but not in MEF Rad23a−/−cells (Figure 3F). When MEF cells were treated withother pro-apoptotic drugs such as actinomycin D,etoposide and cisplatin in the presence of broad-spectrum caspase inhibitor Q-VD-OPH, a simi-lar result was observed, that is AIF was caspase-independently translocated into the nucleus in MEFWT, and MEF Rad23b−/− cells but not in MEFRad23a−/− cells (Supplementary Figure S3). Furtherevidence showed that the Rad23a−/− cells decreasedthe nuclear accumulation of mouse UV excision re-pair protein RAD23 homolog B (mHR23B), and theRad23b−/− cells, however, did not affect the nuclearaccumulation of mouse UV excision repair proteinRAD23 homolog A (mHR23A) (Supplementary Fig-ure S2B). Together, these data support our hypothesisthat hHR23A is required for the nuclear transloca-tion of AIF during induction of cell death.

The XPC binding domain of hHR23A interactswith the N-terminal region of AIFTo identify the binding regions between hHR23Aand AIF, various deletion mutants of the indivi-dual genes were employed. Three deletion mutants,which excluded, C-terminal region of hHR23A andN-terminal region of AIF, were constructed to lo-cate the potential binding segments (Figure 4A). The

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Figure 3 The effect of RAD23A silencing on STS-induced nuclear translocation of AIF and cisplatin-mediated cytotoxicity.(A) To inhibit the hHR23A protein expression, HeLa and H2087 cells were infected with recombinant retroviruses encoding

shRNAs specific to RAD23A (pRS-RAD23A; clones #S1 and #S2), and then selected in the medium containing puromycin

(2 μg/ml). Equal amounts of whole cell lysates were analysed by immunoblotting with anti-hHR23A or anti-AIF antibodies. pRS

vector, pSuper.retro.puro retroviral shRNA expressing vector; pRS-RAD23A S1 & pRS-RAD23A S2, pRS vectors harbouring

RAD23A-gene specific shRNAs. (B) The HeLa wild-type cells, pRS-mock (mock-shRNAs), pRS-RAD23A S1 and RS-RAD23A

S2 infected cells were grown on silane-coated glass slides for 36 h and then treated with or without STS (1 μM for 3 h) and

then fixed. The fixed cells were stained for AIF (red fluorescence), mitochondria (HSP60, green fluorescence) and then counter

stained for nuclei by using DAPI (blue fluorescence). Images were acquired by confocal immunofluorescence microscopy. The

scale bars represent 10 μm. (C) The AIF-positive nuclei were quantified in RAD23A-silenced HeLa cells after treatment with STS

(1 μM for 3 h) and expressed as the percentage of the cells containing AIF-positive nuclei in total counted cells (n � 100). Values

(mean ± s.d.) are from three independent experiments; ***P ˂ 0.001. (D) To quantify the cisplatin-mediated cytotoxicity in RAD23A-

silenced HeLa cells, the cells were treated continuously with cisplatin in a dose-dependent manner for 72 h and then analysed

by cytotoxicity assay. Values (mean ± s.d.) are from three independent experiments. (E) E1, Mouse embryonic fibroblasts (MEFs)

wild-type (WT) cells; E2, Rad23a gene knockout (Rad23a−/−) MEFs; and E3, Rad23b gene knockout (Rad23b−/−) MEFs were

treated with or without STS (50 nM for 18 h) and then fixed. The fixed cells were stained for AIF (red fluorescence) and counter

stained for nuclei by using DAPI (blue fluorescence). The fluorescence intensity in the cells (AIF: red line; DAPI: blue line) was

measured along the white line (the fourth column). Overlap of red line with blue line indicated nuclear translocation of AIF. (F)

The subcellular fractionation and immunoblotting analysis show the level of AIF in the mitochondrial fraction decreased while

that in the nuclear fraction increased following STS (50 nM for 18 h) treatment in MEF WT, and MEF Rad23b−/− cells but not in

MEF Rad23a−/− cells. STS also increased the level of 58-kDa mHR23A in both the mitochondrial and the nuclear fractions of

MEF WT and MEF Rad23b−/− cells.

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Figure 3 Continued.

endogenous AIF in HeLa cells interacted with thehHR23A fragment (phHR23A-N290), which con-tained the amino acid residues 232–283 (Figure 4B),an area that had been suggested to be interactingwith the xeroderma pigmentosum group C (XPC)

protein (Kamionka and Feigon, 2004). Moreover, en-dogenous hHR23A in HeLa cells interacted withthe AIF fragment (pAIF) that included the aminoacid residues 1–100 (Figure 4C). Photographic scan-ning of the electrophoretic images showed that the

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Figure 4 Identification of the binding regions between hHR23A and AIF by using deletion mutants(A) Schematic presentation for the hHR23A and AIF wild-type and their C- or N-terminus deletion mutants with respect to the

location of their protein domains. The wild-type and deletion mutants were constructed into pcDNA3.1TM/myc-His mammalian

expression vector as described in the Materials and Methods. (B) The indicated forms of hHR23A constructs were transiently

expressed as myc-tagged fusion proteins in HeLa cells for 36 h. To examine the binding between mutant hHR23A-fusion proteins

and endogenous AIF, the whole cell lysates (WCL) were incubated with antibodies to myc-tag and the immunocomplexes were

precipitated by using resin-conjugated protein G and resolved by immunoblotting with anti-AIF or anti-myc antibodies (upper

panel). To monitor the expression levels of myc-tagged hHR23A-fusion proteins, an equal amount of WCL was analysed by

immunoblotting with anti-myc antibodies (lower panel). (C) The indicated forms of AIF constructs were expressed as myc-tagged

fusion proteins in HeLa cells. To examine the co-immunoprecipitation of endogenous hHR23A and mutant AIF-fusion proteins,

an equal amount of WCL was incubated with anti-hHR23A or anti-myc antibodies and the immunocomplexes were subjected

to immunoblotting with anti-myc or anti-hHR23A antibodies (upper and middle panel). To monitor the expression levels of myc-

tagged AIF-fusion proteins, an equal amount of WCL was analysed by immunoblotting with anti-myc antibodies (lower panel).

*, indicates the degradation of AIF-myc or mutant AIF-myc fusion proteins; IP, immunoprecipitation; IB, immunoblotting; IgGhc,

immunoglobulin heavy chain; IgGlc, immunoglobulin light chain.

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hHR is required for AIF transport Research article

intensity ratio of protein bands between hHR23Aand AIF was 4:1.

Interaction between hHR23A and AIF was fur-ther studied by using confocal FRET microscopy.The binding fragments of hHR23A, and AIF wereco-expressed as hHR23A (AA207–297)-CFP, andAIF (AA1–100)-YFP fusion proteins in HEK 293cells to detect the FRET signal. The cells express-ing CFP–YFP fusion proteins alone (positive con-trol), when excited at 405 nM wave length, FRETsignal was detected between CFP and YFP proteins(Figure 5A). The cells expressing hHR23A (AA207–297)-CFP and AIF (AA1–100)-YFP fusion proteinsexhibited no FRET signal upon excitation at 405 nMwave length (Figure 5B). Following exposure of thecells to STS, both hHR23A (AA207–297)-CFP andAIF (AA1–100)-YFP fusion proteins were translo-cated to the nuclei, and the interaction betweenhHR23A and AIF became more evident by the de-tection of FRET signal (Figure 5C). The occurrenceof FRET was confirmed by the emission spectrum,which showed, when cells were excited at 405 nMwavelength, the emission intensity was maximum at530 nM wavelength in the positive control and STS-treated cells but not in the untreated control cells(Figure 5D), indicating protein–protein interaction.The FRET efficiency was further confirmed by photo-bleaching, which showed the FRET efficiency dimin-ished dramatically upon photobleaching (Figure 5C,arrow) and the quantitation of apparent FRET effi-ciency indicated that hHR23A interacted with AIF(Figure 5E).

2,4-Dinitrophenol inhibits STS-induced nucleartranslocation of AIF and cisplatin-mediatedcytotoxicityAs noted, after protein synthesis, the AIF is origi-nally targetted to the mitochondria, and the nucleartargetting of AIF is a secondary event. Therefore, theroute and transport vehicles for nuclear targetting ofAIF may differ from those of the primary nucleartargetting factors. The factors other than the rou-tine cytoplasm-nucleus shuttle proteins could existfor AIF and our data suggest that hHR23A could bea prospective candidate. Moreover, because vehicle-mediated nuclear import of macromolecules is energydependent (Schwoebel et al., 2002) and the deple-tion of ATP supply rapidly shuts down the classical

nuclear localisation signal (cNLS)-associated nucleartransport (van der Spek et al., 1996). We, there-fore, examined whether the nuclear translocation ofAIF and cisplatin-mediated cytotoxicity were affectedby the ATP supply. Addition of 2,4-dinitrophenol(DNP) (respiratory ATP synthesis inhibitor) reducedthe STS-induced nuclear translocation of AIF in A549cells (Figures 6A and 6C). Consistently, addition ofDNP reduced STS-induced nuclear translocation ofhHR23A in A549 cells (Figure 6B) and increased cis-platin resistance in H838 and HeLa cells (Figure 6D)as well.

KPNA2 is not essential for STS-induced nucleartranslocation of AIFPrevious studies have shown that karyopherin sub-unit alpha-2 (KPNA2) and karyopherin subunitbeta-1 (KPNB1) heterodimer is the major vehiclewhich transports the proteins with cNLS through thenuclear–pore complexes (NPC) (Chook and Blobel,2001; King et al., 2006; Lange et al., 2007). Initially,KPNA2 recognises and binds to the cNLS of thenuclear-targetting proteins and forms a ternary com-plex shuttle (TCS) with KPNB1 to pass through theNPC and enter the nucleus. Therefore, silencing theKPNA2 (NCBI Reference Sequence: NM 002266.2)protein expression would then inhibit the nuclearimport of the proteins with cNLS. However, ourdata shows, silencing the KPNA2 protein expres-sion in H838 cells (Figure 7A) did not affect theSTS-induced nuclear transport of AIF, indicating thatKPNA2 was not involved in the nuclear transloca-tion of AIF (Figures 7B and 7C).

DiscussionThe present results show that the nuclear transloca-tion of AIF requires hHR23A.

The hHR23A and hHR23B are human homologsof S. cerevisiae Rad23 (Sugasawa et al., 1997). Boththe proteins have been proposed to play crucial rolesin the nucleotide excision repair (NER) mechanism.However, the appearance of facial disfigurement, andthe male infertility exclusively in the Rad23b−/−,but not in the Rad23a−/− mice indicates that thehHR23A, and the hHR23B may physiologicallyact differently (Rockett et al., 2004). The fact thathHR23A is recruited to the mitochondria, and bothAIF and hHR23A are rapidly accumulated inside the

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Figure 5 Detection of hHR23A and AIF interaction by using FRET(A) To detect the FRET signal, CFP-YFP fusion protein was used as a positive control. Plasmid encoding CFP and YFP was

transiently expressed as CFP-YFP fusion protein in HEK 293 cells for 36 h and then fixed. The fixed cells were excited at

405 nM wave length for FRET occurrence (white circle areas). (B) To detect the FRET signal between hHR23A and AIF, se-

quences corresponding to amino acids 207–297 (AA207–297) of hHR23A and amino acids 1–100 (AA1–100) of AIF were inserted

N-terminally into pECFP and pEYFP mammalian expression vectors. The indicated constructs were transiently co-expressed

as hHR23A (AA207–297)-CFP and AIF (AA1–100)-YFP fusion proteins in HEK 293 cells for 36 h. The white circle areas indicate

no FRET signal detection upon excitation at 405 nM in the control cells. (C) The HEK 293 cells expressing hHR23A (AA207–

297)-CFP and AIF (AA1–100)-YFP fusion proteins were treated with 1 μM STS for 3 h to detect the protein–protein interaction.

The white circle areas indicate FRET occurrence upon excitation at 405 nM. (D) The above cells of the panel A, positive control;

B, untreated control cells; and C, STS-treated cells were excited at wavelength 405 nM (ECFP excitation) and the emission

spectrum was observed at 430–600 nM wavelength (EYFP emission) respectively to detect the FRET signal. Arrow indicates the

maximum emission intensity (530 nM). (E) Quantitation of the FRET efficiency as detected by photobleaching the acceptor AIF

(AA1–100)-YFP fusion protein at 514 nM wave length before excitation of the donor hHR23A (AA207–297)-CFP fusion protein in

the cells of Fig.5C (arrow indicates photobleaching).

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Figure 6 The effect of DNP on STS-induced nuclear translocation of AIF and cisplatin-mediated cytotoxicity(A and B) A549 cells were treated with or without STS (1 μM for 3 h), or 2,4-dinitrophenol (DNP) (5 mM for 3 h), or both and

then fixed. The fixed cells were stained for AIF (red fluorescence), or hHR23A (red fluorescence) and counter stained for nuclei

by using DAPI (blue fluorescence). The scale bars represent 10 μm. (C) AIF-positive nuclei were quantified after the A549 cells

were treated with or without STS, or DNP, or both and expressed as the percentage of the cells containing AIF-positive nuclei

in total counted cells (n � 100). Values (mean ± s.d.) are from three independent experiments; ***P ˂ 0.001. (D) H838 and HeLa

cells were incubated with or without DNP (5 mM for 3 h) and then treated with cisplatin (50 μm for 72 h). The percentage of cells

survived was quantified by cytotoxicity assay. Values (mean ± s.d.) are from three independent experiments.

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Figure 7 The effect of KPNA2 silencing on STS-induced nuclear translocation of AIF(A) To inhibit the karyopherin subunit alpha-2 (KPNA2) protein expression, H838 cells were infected with recombinant lentiviruses

encoding shRNAs specific to KPNA2 (pLKO.1-KPNA2; clones #1 and #2), or vector alone (pLKO.1-puro) and then selected in

the medium containing puromycin (2 μg/ml). Equal amounts of whole cell lysates were analysed by immunoblotting with the

indicated antibodies. (B) KPNA2-silenced H838 cells were treated with or without STS (1 μM for 3 h) and then fixed. The fixed

cells were stained for AIF (red fluorescence) and counter stained for nuclei by using DAPI (blue fluorescence). The scale bars

represent 10 μm. (C) AIF-positive nuclei were quantified in KPNA2-silenced H838 cells after treatment with or without STS and

expressed as the percentage of the cells containing AIF-positive nuclei in total counted cells (n � 100). Values (mean ± s.d.) are

from three independent experiments.

nuclei following genotoxic stress strongly suggeststhat AIF and hHR23A indeed interact with eachother. The deletion of Rad23a gene in MEFs, and si-lencing the hHR23A protein expression in HeLa cellsnot only reduced the nuclear level of AIF, but also re-duced cisplatin-induced cytotoxicity further supports

our hypothesis and confirms that AIF plays a criticalrole in mediating the drug-related apoptosis.

Several studies have shown that AIF is a po-tential intracellular target for clinically importantanticancer agents, for example topoisomerase IIinhibitors (epipodophyllotoxins and intercalating

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agents), topoisomerase I inhibitor (camptothecin) andDNA alkylating agents (cisplatin). Recent evidencessuggested that in addition to the direct interac-tion with DNA and DNA-associated enzymes (Chowet al., 1988), these drugs induce mitochondria-related cell death through the activation of caspasesand the nuclear translocation of AIF as well (Brus-tugun et al., 1998; Daugas et al., 2000; Susin et al.,2000; Joseph et al., 2002; Vahsen et al., 2004; Che-ung et al., 2006). Although the function of AIF in-side the nucleus is yet to be clarified, the intranuclearevents that follow AIF translocation are critical forcytotoxicity to take place. The present study shedsfurther light on the role of hHR23A during nu-clear translocation of AIF and how this may affectthe drug sensitivity. The simultaneous reduction inAIF nuclear translocation an cisplatin sensitivity inthe RAD23A-gene silenced HeLa cells clearly in-dicate that hHR23A affects the nuclear transportof AIF and drug sensitivity. Increase of RAD23Alevels in the mitochondria following STS treatmentin A549, HeLa, MEF WT and MEF Rad23b−/−cells suggested that the protein was recruited tothe mitochondria during the initiation of apoptosis.Therefore, like AIF, hHR23A could also be rapidlyaccumulated inside the nuclei following genotoxicstress. Decrease in hHR23A levels not only restrainsthe nuclear transport of AIF, but also reduces thecisplatin-mediated cytotoxicity. These observationsimplicate that hHR23A besides protein degradation(van der Spek et al., 1996) and DNA repair (Swederand Madura, 2002) could also facilitate the nucleartranslocation of AIF to initiate nuclear apoptosis.

Previous studies have shown that KPNA2 andKPNB1 heterodimer is the major vehicle that trans-ports proteins with cNLS through the NPC (Chookand Blobel, 2001; King et al., 2006; Lange et al.,2007). Exportin-CAS, on the other hand, recycleskaryopherins back to the cytoplasm when the nu-clear cargo is unloaded from the TCS by RanGTP(Schwoebel et al., 2002; King et al., 2006). However,our data show that karyopherins neither interactedwith AIF in the yeast two-hybrid assay nor they co-precipitated with the antibodies specific to AIF inA549 cells. Moreover, silencing the KPNA2 pro-tein expression in H838 cells did not affect the nu-clear translocation of AIF after STS challenge. Thesedata indicate that in addition to karyopherins (Kinget al., 2006), other proteins could be involved in

the transportation of AIF into the nucleus follow-ing genotoxic challenge. Furthermore, the nucleartranslocation of AIF, being a secondary organelle tar-getting, may adapt a different route other than thekaryopherins. The fact that the presence of hHR23Ain both the cytoplasm and the nucleus (van derSpek et al., 1996) further support our data thathHR23A is clearly involved in the nuclear proteintransport.

As shown earlier that by using a yeast two-hybridassay to search for AIF binding proteins, we had iden-tified hHR23A, Microsomal GST-1 and CASP-14. Ina previous study, we had shown that caspase-14 in-hibited the apoptotic function by directly bindingto the AIF (Fang et al., 2011). Moreover, we foundthat hHR23A could facilitate the nuclear transportof dynamin-related protein 1 to particularly pro-tect genomic DNA in the nucleoli (Chiang et al.,2009). Thus, it is worth noting that hHR23A in-teracts with the viral protein R (Vpr) of human im-munodeficiency virus (HIV) (Huang et al., 2012).The Vpr, a 14-kDa protein composed of 96 aminoacids, is essential for the nuclear transport of pre-integration complex (PIC) and intranuclear replica-tion of HIV. In vitro, heat shock protein 70 (HSP70)could replace the Vpr for nuclear importation of PIC(Agostini et al., 2000). Interestingly, HSP70 couldalso bind to AIF, and inhibit the nuclear transportof AIF to reduce the cytotoxic effect of anticancerdrugs (Ravagnan et al., 2001; Gabai et al., 2005).Silencing the hHR23A protein expression increasedp53 degradation and suppressed apoptosis, whereasenforced expression of hHR23A induced apoptosis(Gaynor and Chen, 2001; Brignone et al., 2004; Kauret al., 2007). These findings support our present data,and our results suggest that the effect of hHR23Aon apoptosis is through AIF. The results from dele-tion mutants and FRET study further indicatedthat the motif NPALLPALLQQLGQENPQLLQQ[amino acid residues (aar) 251–271] in hHR23Acould be the area that interacted with the MASS-GASGGKIDNSVLVLIVGLSTVGAGAYA (aar 53–84) motif of AIF. The weak signal from FRET be-tween hHR23A and AIF prior to the initiation ofapoptosis suggested that the two proteins were phys-ically separated during protein synthesis, and the ini-tial mitochondrial importation of the AIF in the cells(Chiang et al., 2012). Therefore, the two proteinscan encounter with each other only when the AIF is

C© 2014 Societe Francaise des Microscopies and Societe de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd 371

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released from the mitochondria following apopto-genic challenge.

Unlike what has been reported previously thatshowed that the mouse AIF has three forms: a 67-kDa full-length precursor, a 62-kDa mature form anda 57-kDa apoptotic form (Otera et al., 2005); here, wedetected only a 67-kDa AIF in the human cancer cells.We have checked 26 human cancer cell lines that wereavailable, and the major form we found was indeed67-kDa AIF (Supplementary Figure S4). By usingIP assay and two-dimensional gel electrophoresis, weisolated and characterised the AIF protein from thehuman cancer cells and its identity was determinedby using MALDI-TOF. All these results showed thatAIF protein in the human cancer cells is 67-kDa full-length form with intact N-terminus region (Chenet al., 2006; Chen et al., 2008).

It remains to be elucidated that neither AIF norhHR23A per se possesses any nuclease activities, buttheir presence in the nuclei is closely associated withthe DNA fragmentation. It is possible that whenthe cells are challenged with anticancer reagents,the DNA strand breakage is transiently introducedinto the drug-stabilised ‘cleavable-complexes’, ren-dering free 3′-ends of DNA (Chow and Ross, 1987).The concurrent rush-in nuclear AIF and hHR23Acould initiate and accelerate the DNA degradationat strand breakage sites when the drug-related DNAdamage was not repaired on time (Brustugun et al.,1998; Susin et al., 2000; Joseph et al., 2002). Since,the drug-induced DNA damage and cytotoxicity areATP dependent (Chow and Ross, 1987; Chow et al.,1988; Hatta et al., 2004), it is not surprising to findthat the treatment with DNP to reduce intracellu-lar ATP level decreases drug cytotoxicity (Brignoneet al., 2004), and the addition of ATP increases thecell sensitivity to etoposide (Hatta et al., 2004). Ourresults corresponded well with those data, and showedthat the nuclear translocation of AIF was energy de-pendent. Based on the DNA homology with yeastRad23, hHR23A has been suggested to play a role inthe NER, a mechanism that is closely associated withdrug resistance (Viktorsson et al., 2005). This is con-sistent with our findings that the levels and activitiesof hHR23A and AIF are coupled in determining drugsensitivity. Our data further show that hHR23A fa-cilitates the nuclear translocation of AIF, events thatare essential for the subsequent nuclear changes anddrug sensitivity of the cells. In conclusion, by show-

ing that AIF and hHR23A interact with each other,and silencing the hHR23A protein expression de-creases the nuclear translocation of AIF and cisplatinsensitivity, our results suggest that hHR23A is re-quired for the nuclear transport of AIF during theinitiation of cell death.

Materials and methodsChemicals and antibodiesSTS (569396), actinomycin D (114666) and etoposide (341205)were purchased from Calbiochem. Cisplatin (3078-20) waspurchased from Bristol-Myers Squibb. Mouse anti-β-actin(A5441), mouse anti-α-tubulin (T6074) antibodies and DNP(40057) were purchased from Sigma–Aldrich. 4′,6-Diamidino-2-phenylindole (DAPI) (H3570), Prolong Gold AntifadeReagent (P36934), and rabbit anti-COX IV (4850), rabbit anti-Histone H3 (9715), rabbit anti-HSP60 (12165) primary an-tibodies and goat anti-rabbit or anti-mouse conjugated withAlexa Fluor 488 (A11008) or 546 (A11003) secondary antibod-ies were purchased from Invitrogen Life Technologies. Mouseanti-KPNA2 (610486) antibody was purchased from BD Trans-duction Laboratories. Mouse anti-TOM20 (sc-17764) and mouseanti-c-Myc (sc-40) were purchased from Santa Cruz Biotech-nology. Horseradish peroxidase (HRP)-labelled goat anti-mouseIgG (115-036-003) or anti-rabbit IgG (111-035-003) were pur-chased from Jackson Immuno Research Laboratories.

Cell lines and cell cultureSeven lung adenocarcinoma (ADC) cell lines (H23, H226, H838,H1437, H2009, H2087 and A549) were used for the in vitrostudies (Chen et al., 2006). Cells were maintained at 37 °C asa monolayer in RPMI-1640, or DMEM for HeLa, HEK 293and HEK 293T cells supplemented with 10% foetal calf serum,100 IU/ml of penicillin and 100 μg/ml of streptomycin in ahumidified 5% CO2 incubator.

RNA extraction, reverse transcription PCR and preparationof monoclonal antibodiesTotal RNA was extracted from lung ADC cell lines by using anSNAP RNA column (Invitrogen). After measuring total RNAyield, cDNA was synthesised by AMV reverse transcriptase usingoligo random primers. An aliquot of cDNA was then subjectedto 35 cycles of PCR. The reaction mixture contained 50 mMTris (pH 7.2), 1.5 mM MgCl2, 2 μM dNTP, 0.25 μM of 5′ and3′ primers, respectively, 0.5 U of Taq DNA polymerase (ProtechTechnology) and 2 μl of cDNA (1 μg). PCR was carried outaccording to the standard procedures: denaturing at 94 °C for30 s, hybridising at 55 °C for 45 s and elongating at 72 °C for2 min.

The primer sequences for AIF were (forward primer) 5′-ATGTTCCGGTGTGGAGGCCTGGCGGCGGGTG-3′ (nts232–262, NM 004208.3) and (reverse primer) 5′-TCAGTCTTCATGAATGTTGAATAGTTTGGCT-3′ (nts 2073-2043); and those for RAD23A were (forward primer) 5′-GCCatgGCCGTCACCATCACGCTCAAAACGCTG-3′ (nts133–165, NM 005053.3) and (reverse primer) 5′-

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TTCCTGGCAtcaCTCGTCATCAAAGTTCTGACT-3′ (nts1236-1204).

The amplified PCR products were analysed in 1% agarose gel,and visualised by ethidium bromide staining. The full-lengthcDNA fragment of AIF was 1842 base-pairs (bp), and thatof RAD23A was 1104 bp. The respective fragments were geleluted and cloned into pCR

R©2.1-TOPO vector (Invitrogen

Life Technologies). The DNA sequence corresponding to theC-terminal amino acids 315 to 612 of AIF, and the full-lengthhHR23A were further amplified by PCR using pCR

R©2.1-

TOPO-AIF, and pCRR©2.1-TOPO-RAD23A as the templates

with primer sequences containing either EcoR I (sense) or Not I(antisense) restriction sites for AIF, and the primer sequencescontaining either Hind III (sense) or Xho I (antisense) restrictionsites for RAD23A. The primer sequences for AIF were (forwardprimer) 5′-TCCGAATTCATGCTGGCCTGTGCTCTTGGC-3′ (nts 1174–1191, EcoR I site is underlined) and (reverse primer)5′-CGTGCGGCCGCATCAGTCTTCATGAATGTTGA-3′(nts 2073-2054, Not I site is underlined); and those for RAD23Awere (forward primer) 5′-CGACAAGCTTGCATGGCCGTCACCATCACGCTC-3′ (nts 136–156, Hind III site is under-lined) and (reverse primer) 5′-TGGTGCTCGAGTTCACTCGTCATCAAAGTTCTG-3′ (nts 1227-1207, Xho I site is under-lined). The initiation and stop codons are shown in bold-phasealphabets.

The cDNA fragments of AIF and hHR23A were then insertedin frame into the polylinker region of the bacterial expressionvector pET-32a+ (Promega KK). A bacterial colony containingplasmid with the correct gene sequence was selected, and in-duced to mass-produce recombinant proteins. The recombinantproteins were isolated by a Nickel-affinity column, and the pro-tein identity was determined by MALDI-TOF. Affinity-purifiedproteins were used to immunise BALB/c mice (6–8-week old).Sensitivity and specificity of the antisera were respectively de-termined by ELISA (OD405 > 0.5 at 1:6000 dilutions) andimmunoblotting (antisera, 1:3000 dilution). Monoclonal anti-bodies were produced by a cell fusion hybridoma technique andscreened by the afore-mentioned methods.

IP, gel electrophoresis, immunoblotting and proteinanalysis by MALDI-TOFThe procedure for IP was described previously (Chen et al.,2008) with minor modifications. Briefly, total cell lysates wereprepared by mixing 5 × 107 cells/ml PBS with equal volumeof 2 × NP-40 lysis buffer [40 mM Tris–HCl, pH 7.6, 2 mMEDTA, 300 mM NaCl, 2 mM PMSF and 2% NP-40, supple-mented with leupeptin (20 μg/ml), aprotinin (20 μg/ml) andtrypsin inhibitor (20 μg/ml)]. Protein G sepharoseTM (Amer-sham Biosciences AB) was washed with NP-40 lysis buffer twicebefore mixing with 5 μg of purified monoclonal antibodies.The reaction mixture was incubated at 4 °C with constant ro-tation for 60 min, and then centrifuged at 800g for 1 min.The pellet was re-suspended with 500 μg of total cell lysate,and incubated at 4 °C with constant rotation for 18 h beforecentrifugation at 800g for 1 min. Following removal of thesupernatant, the precipitate was washed with 1 × PBS threetimes, and then dissolved in loading buffer (50 mM Tris, pH6.8, 150 mM NaCl, 1 mM disodium EDTA, 1 mM PMSF, 5%β-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue

and 1% SDS). For low stringent wash, the reaction mixturewas washed with PBS containing 100 mM NaCl twice beforebeing dissolved in loading buffer. Electrophoresis was carriedout in two 10% polyacrylamide gels with 4.5% stacking. Onegel was stained with Coomassie Brilliant blue, and the othergel was processed for immunoblotting (Chow and Ross, 1987).For immunoblotting, the proteins were electro-transferred toa polyvinylidene fluoride membrane after electrophoresis. Themembrane was probed using specific antibodies followed byincubation with the appropriate HRP-labelled secondary anti-bodies. The protein bands were detected using an immobilonwestern chemiluminescent HRP substrate (Merk Millipore) anddeveloped using autoradiography (Fujifilm Corporation). Thedigital images were processed using Adobe Illustrator CS6(Adobe Systems Incorporated). The protein band on Coomassie-stained gel that corresponded to the immunoblot-positive bandwas extracted from the gel for identification by MALDI-TOFon a Voyager-DETM pro biospectrometry workstation (Ap-plied Biosystems). Sequences of the oligopeptide fragmentswere matched with those on the SwissProt database by MS-fit (ProteinProspector 4.0.5.; The Regents of the University ofCalifornia).

Cytotoxicity assayCytotoxicity was determined by a modified MTT method, inwhich activity of the mitochondrial dehydrogenase was usedas a measure (Chen et al., 2006). Cells were seeded at 1000,2500, 5000, and 10,000 cells/well in a 96-well plate 18 h priorto drug challenge. The cells were then treated continuouslywith various concentrations of cisplatin (ranged from 1.6 μMto 1.0 mM) for 72 h. The control group was treated with PBSonly. Following drug challenge, 10 μl of WST-1 (BioVision)was added and the cells were allowed to incubate for 2 h; theplates were read at 450 nM optical density. The percentage ofcells that survived in the treatment group was quantified bycomparing with the number of cells that survived in the controlgroup. All procedures were performed in triplicate. The effectof ATP content on drug sensitivity was analysed by incubatingcells with various concentrations of DNP for 1 h before challengewith cisplatin.

Silencing the RAD23A and KPNA2 gene expression byusing small interfering RNATo silence the RAD23A gene expression, recombinant retro-viruses encoding siRNAs specific to RAD23A (pRS-RAD23A;clones #S1 and #S2) were used. The target sequences forRAD23A (NM 005053.3) siRNA were 5′-GGUGCUAAAGGAGAAGAUA-3′ (nts 213–231) and 5′-AGGAGAAGAUAGAAGCUGA-3′ (nts 221–239). Double-stranded oligonu-cleotides containing the following shRNA (short-hairpinRNA) sequences: S1, 5′-GATCCCCGGTGCTAAAGGAGAAGATATTCAAGAGATATCTTCTCCTTTAGCACCTTTTTA-3′; and S2, 5′-GATCCCCAGGAGAAGATAGAAGCTGATTCAAGAGATCAGCTTCTATCTTCTCCTTTTTTA-3′ were re-spectively cloned into the BglII and HindIII site of the retroviralvector pSuper.retro.puro vector (pRS-puro) (OligoEngine). Plas-mid containing 5′-GATCCCCCTACCGTTGTTATAGGTGTTTCAAGAGAACACCTATAACAACGGTAGTTTTTA-3′shRNA was used as mock control. To produce recombinant

C© 2014 Societe Francaise des Microscopies and Societe de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd 373

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retroviruses, gp 293 cells were co-transfected with pRS-RAD23A; clones #S1 or #S2 (4.5 μg) and pVSV-G (0.5 μg) byjetPEITM DNA transfection reagent (Polyplus transfectionTM)in a 6 cm dish. After 72 h, the medium was harvested, aliquotedand stored at −80 °C. To silence the RAD23A gene expressionin HeLa cells, they were infected with recombinant retrovirusesencoding RAD23A specific siRNAs with an MOI >6 inthe presence of 4 μg/ml polybrene for 24 h. The cells wererinsed in DMEM and allowed to grow for another 36 h in thegrowth medium. Subsequently, the cells were selected in thegrowth medium containing 2 μg/ml of puromycin (TOCRISbioscience) for 1 week. The puromycin-resistant HeLa cells werecollected and the RAD23A gene knockdown in those cells wasdetermined by immunoblotting with anti-hHR23A antibody.

To inhibit the KPNA2 protein expression, recombi-nant lentiviruses encoding siRNAs specific to KPNA2(pLKO.1-KPNA2; clones #1 and #2) were used. The tar-get sequences for KPNA2 (NM 002266.2) siRNA were 5′-CTACCTCTGAAGGCTACACTT-3′ (nts 1661–1681) for clone#1, and 5′-TCATGTAGCTGAGACATAAAT-3′ (nts 1724–1744) for clone #2. The above-mentioned sequences were re-spectively cloned into the EcoRI site of the lentiviral vectorpLKO.1-puro. To produce recombinant lentiviruses, HEK 293Tcells were co-transfected with vector alone or pLKO.1-KPNA2(2.5 μg), pCMV-�R8.91 (2.25 μg) and pMD.G (0.25 μg) byjetPEITM DNA transfection reagent in a 6 cm dish. After 72 h,the medium was harvested, aliquoted and stored at −80 °C. Tosilence the KPNA2 gene expression in H838 cells, they were in-fected with recombinant lentiviruses encoding KPNA2 specificsiRNAs with an MOI >6 in the presence of 8 μg/ml polybrenefor 24 h. The cells were selected in the growth medium contain-ing 2 μg/ml of puromycin for 5 days. The puromycin-resistantH838 cells were collected and the KPNA2 gene knockdownin those cells was determined by immunoblotting with anti-KPNA2 antibody.

Fractionation of cellular componentsFractionation of subcellular components was per-formed according to Calbiochem instructions(http://www.merckbiosciences.co.uk) with minor modifi-cations. The cells were detached from culture plates by treatingwith dissociation buffer (Sigma) at 37 °C for 2–5 min. Afterwashing with PBS, cells were re-suspended in homogenisationbuffer [10 mM Tris–HCl, pH 7.4, 10 mM KCl, 1.5 mM MgCl2,1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM dithiothreitol,250 mM sucrose, 0.1 mM PMSF, leupeptin (10 μg/ml),aprotinin (10 μg/ml) and trypsin inhibitor (10 μg/ml)] at 4 °Cfor 15 min. After 80 strokes with a B pestle in a Douncer, theunbroken cells were removed by centrifugation at 100g for5 min. The nuclei were collected by spinning the solution at1000g for 10 min, and the mitochondria were collected fromthe supernatant by centrifugation at 11,000g for 10 min. Aftercentrifugation at 20,000g for 20 min to remove the insolubleresidue, the final supernatant was used as cytosolic fraction.

Immunofluorescent stainingFor immunofluorescence staining, the indicated cell lines in thestudy were grown on silane coated glass slides for 36 h and thenfixed with 4% formaldehyde at room temperature for 15 minand then permeabilised using 0.3% Triton X-100. After wash-

ing three times with PBS, the cells were incubated with theprimary antibodies for 90 min, and washed three times withPBS. After the primary antibodies were washed off, the slideswere incubated with Alexa flour 488 or 546-conjugated sec-ondary antibodies. The nuclei were counter stained by usingDAPI. A cover slip was mounted on the cells by using Pro-long Gold Antifade Reagent. The slides were examined undera laser scanning confocal microscope (Olympus, FV1000, Con-focal Laser Scanning Biological Microscope). The images of thecells were analysed by using FV10-ASW 3.1 software and AdobePhotoshop CS6 (Adobe Systems Incorporated).

Construction of deletion mutants for AIF and hHR23AThe full-length AIF was amplified by PCR using pCR

R©2.1-

TOPO-AIF as the template with forward primer 5′-CGAGCGGCCGCCATGTTCCGGTGTGGAGGCCTG-3′ (nts232–252) and reverse primer 5′-TGGAATTCTGCAGTCTTCATGAATGTTGAATAG-3′ (nts 2070-2050) andcloned in frame to the Not I and EcoR I site (underlinedin the primer sequence) of pcDNA3.1TM/myc-His (A)vector (Invitrogen Life Technologies). The construct wasnamed as pAIF which encoded 613 amino acids (a.a.). TheN-terminus deletion mutants of AIF were created by PCRusing pCR

R©2.1-TOPO-AIF as the template with different

forward primers and the reverse primer was the same one forall the deletion mutant constructs. The fragments were clonedin frame to the Not I and EcoR I site of pcDNA3.1TM/myc-His(A) vector. To construct pAIF �N100 (deletion of sequencecorresponding to a.a. residues 1–100), the forward primer 5′-CGAGCGGCCGCCATGGGGTTAGGGCTGACACCAGAA-3′ (nts 532–552); similarly, to construct pAIF�N122 (deletion of sequence corresponding toa.a. residues 1–122), the forward primer 5′-CGAGCGGCCGCCATGGTTCCTCAAGACAAGGCGCCA-3′ (nts 598–618); and to construct pAIF �N263 (deletion of se-quence corresponding to a.a. residues 1–263), the forward primer5′-CGAGCGGCCGCCATGCCAAGAAGTCTGTCTGCC-3′(nts 1021–1038) were used respectively. The Not I site isunderlined and the initiation codon is shown in bold-phasealphabets in the forward primer sequences.

The C-terminus deletion mutants of hHR23A werecreated by PCR using pCR R©2.1-TOPO-RAD23A as thetemplate. To construct phHR23A N85 (deletion of sequencecorresponding to a.a. residues 86–363), the reverse primer 5′-CCCAAGCTTGGTGGTACCCTGGCCGGCTTTGGT-3′ (nts390-370); to construct phHR23A N210 (deletion of sequencecorresponding to a.a. residues 211–363), the reverse primer5′-CCCAAGCTTGGTGTGTTCCGGCTCGGGGCTCCC-3′ (nts 765-745); and to construct phHR23AN290 (deletion of sequence corresponding toa.a. residues 291–363), the reverse primer 5′-CCCAAGCTTGGTCTCCCCAGGGGGCTCGTTCAG-3′ (nts1005–985) were used respectively. The Hind III site is under-lined in the reverse primer sequences. A common forward primer5′-GCCCTCTAGACTATGGCCGTCACCATCACGCTC-3′(nts 136–156) was used for all the above deletion mutantconstructs. The Xba I site is underlined and the initiationcodon is shown in bold-phase alphabets in the forward primersequence. The fragments were cloned in frame to the Xba I andHind III site of pcDNA3.1TM/myc-His (A) vector.

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Fluorescence Resonance Energy TransferThe respective binding fragment of AIF and hHR23A was clonedinto plasmids (pEYFP and pECFP). The plasmids were deliv-ered into HEK 293 cells by cationic polymers. The resultingcells were examined by a confocal microscope, Leica TCS SP5(Leica Microsystems), with excitation at 405 nM (ECFP), andobservation at 430–600 nM wave length.

Author contributionJ.N.S. performed cell culture, manipulation, West-ern blots, siRNA/viral transfection, confocal im-munofluorescence microscopy, FRET, data analysisand manuscript preparation; K.C. helped with per-ception of the idea, experimental design, guidance,manuscript preparation and writing.

FundingThis study was supported by grants from the NationalScience Council, Taipei, Taiwan (NSC102-2320-B-005-006), and the Department of Health, ExecutiveYuan, Taipei, Taiwan to the China Medical Univer-sity Hospital, the Cancer Research for Excellence pro-gram (DOH102-TD-C-111-005), Taichung, Taiwan,R.O.C.

AcknowledgementsThe RNAi used for silencing KPNA2 gene expres-sion was obtained from the National RNAi CoreFacility, Molecular Biology/Genomic Research Cen-tre, Academia Sinica, Taipei, Taiwan and supportedby the National Research Program for GenomicMedicine Grants of NSC (NSC 97-3112-B-001-016).

Conflict of interest statementThe authors have declared no conflict of interest.

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Received: 12 February 2014; Accepted: 16 July 2014; Accepted article online: 23 July 2014

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Supplementary Figure Legends

Supplementary Figure S1. The effect of RAD23A silencing on STS-induced subcellular distribution of hHR23A and AIF in HeLa cells. The hHR23A protein expression in HeLa cells was inhibited as described in the Figure 3A legend. The subcellular fractionation and immunoblotting analysis shows the level of AIF in the mitochondrial fraction decreased while that in the nuclear fraction increased following STS (1 µM for 3 h) treatment in wild type, and pRS-mock infected cells but not in RAD23A silenced cells. STS also increased the level of 58-kDa hHR23A in both the mitochondrial and the nuclear fractions of wild type, and pRS-mock infected cells.

Supplementary Figure S2. Characterization of the monoclonal antibodies to mHR23A and mHR23B. (A) Specificity of the monoclonal antibodies was determined by immunoblotting analysis of the whole cell lysates from the mouse embryonic fibroblasts (MEF) wild type (WT) cells, Rad23a gene knockout (Rad23a-/-) MEFs, and Rad23b gene knockout (Rad23b-/-) MEFs. The 64-kDa protein corresponding to the anticipated molecular mass of mouse UV excision repair protein RAD23 homolog B (mHR23B) is recognized in the whole cell lysates of WT, and Rad23a-/- MEFs. Similarly, the 58-kDa protein corresponding to the anticipated molecular mass of mouse UV excision repair protein RAD23 homolog A (mHR23A) is recognized in the whole cell lysates of WT, and Rad23b-/- MEFs. (B) The MEF wild type cells, Rad23a-/- MEFs, and Rad23b-/- MEFs were treated with or without STS (50 nM for 18 h) and then fixed. The fixed cells were stained for mHR23A/B (red fluorescence) and then counter stained for nuclei by using DAPI (blue fluorescence). In the MEF wild type cells, STS-induced nuclear translocation of mHR23A/B was identical to that found in the A549 cells (Figure 2D). Notably, the Rad23a gene knockout decreased the nuclear accumulation of mHR23B, and the Rad23b gene knockout, however, did not affect the nuclear accumulation of mHR23A.

Supplementary Figure S3. The effect of caspase inhibitor Q-VD-OPH on drug-induced nuclear translocation of AIF in MEF cells. The confocal immunofluorescence microscopic images of MEF wild type, MEF Rad23a-/-, and MEF Rad23b-/- control cells shows AIF (red fluorescence) localization. Actinomycin D (5 µg/mL for 24 h), Etoposide (50 µM for 24 h), and Cisplatin (25 µM for 24 h) treatment in the presence of broad-spectrum caspase inhibitor Q-VD-OPH (50 µM) did not affect the nuclear translocation of AIF in MEF wild type, and MEF Rad23b-/- cells, whereas MEF Rad23a-/-

cells did not show any significant nuclear translocation of AIF. The nuclei were counterstained by using DAPI (blue fluorescence). The scale bars represent 10 µm.

Supplementary Figure S4. The AIF protein expression pattern in the mouse organs and several human cancer cell lines. (A) Except in the mouse kidney, the major form of AIF detected in the mouse organs and the human lung adenocarcinoma cell lines is 67-kDa full-length AIF. The left two lanes show 67-kDa and 57-kDa recombinant AIF protein, respectively. (B) Except in the mouse embryonic fibroblasts (MEFs) cells, the major form of AIF detected in several human cancer cell lines is mainly the 67-kDa full-length AIF. The 57-kDa AIF could be the degraded form of full-length AIF, instead of the apoptotic form.