Identification of ARNT-regulated BIRC3 as the target factor in cadmium renal toxicity

Cadmium (Cd) is an environmental contaminant that exhibits renal toxicity. The target transcription factors involved in Cd renal toxicity are still unknown. In this study, we demonstrated that Cd decreased the activity of the ARNT transcription factor, and knockdown of ARNT significantly decreased the viability of human proximal tubular HK-2 cells. Microarray analysis in ARNT knockdown cells revealed a decrease in the expression of a number of genes, including a known apoptosis inhibitor, BIRC3, whose gene and protein expression level was also decreased by Cd treatment. Although the BIRC family consists of 8 members, Cd suppressed only BIRC3 gene expression. BIRC3 is known to suppress apoptosis through the inhibition effect on caspase-3. Knockdown of BIRC3 by siRNA as well as Cd treatment increased the level of active caspase-3. Moreover, knockdown of BIRC3 not only triggered cell toxicity and apoptosis but also strengthened Cd toxicity in HK-2 cells. Meanwhile, the activation of caspase-3 by suppression of BIRC3 gene expression was mostly specific to Cd and to proximal tubular cells. These results suggest that Cd induces apoptosis through the inhibition of ARNT-regulated BIRC3 in human proximal tubular cells.

The transcriptional regulation of eukaryotic genes involves the organized assembly of multi-protein complexes on promoter regions 19,20 . However, the upstream pathways that regulate gene transcription are controlled by specific regulatory mechanisms; furthermore, not all genes are induced at the same time and with the same duration. Some genes, such as those responsible for correct protein folding, are immediately induced for transcription within minutes; whereas others, such as those involved in DNA damage repair and cell metabolism, are slowly responded to upstream inductions signaling, within hours 21 . Once activated, transcription factors bind to gene regulatory elements (cis-elements), and through interactions with co-factors of the transcription machinery, promote access to DNA and facilitate the recruitment of the RNA polymerase enzymes to the transcriptional start site 19,22 .
Previous studies have shown that Cd exposure enhances the activities of transcription factors that induce cellular protection pathways. For instance, metal response element (MRE)-binding transcription factor-1 (MTF-1) is activated by Cd and induces transcription of MTs [23][24][25] . The Nrf2 transcription factor is also reported to play a role in Cd exposure-mediated gene expression of MT 26 . Although several transcription factors involved in MT expression have been identified, the transcription factors that control the expression of genes associated with Cd renal toxicity are poorly understood.
In this study, we screened transcription factors whose activities were changed by Cd treatment in HK-2 human proximal tubular cells. Among the transcription factors affected by Cd, we investigated the Cd-targeted transcription factors essential for Cd-induced renal toxicity. Furthermore, we examined the downstream factors of Cd-targeted transcription factors that are involved in Cd-induced renal toxicity.

Identification of the transcription factors with altered binding activity in response to Cd treatment in HK-2 cells.
To investigate Cd-induced cytotoxicity in HK-2 cells, we first examined cell viability of HK-2 cells treated with various concentrations of Cd using the MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay (Fig. 1a). HK-2 cells treated with 40 µM Cd for 6 h showed 50% cell toxicity and exhibited less than 10% cell viability at 24 h; however, at 3 h, 40 µM Cd treatment did not induce cytotoxicity. Therefore, we selected these conditions, in which cells remain viable even with Cd treatment (40 µM for 3 h), to examine Cd activation-changed transcription factors using protein/DNA binding arrays in nuclear extracts 27 . Figure 1b shows confirmation and purity of our nuclear extraction process, in which the nuclear marker Lamin A/C is detected in nuclear fractions from both control and Cd-treated cells. Nuclear extracts from control and Cd-treated cells were then incubated with a mixture of 345 biotin-labeled DNA probes corresponding to transcription factor response elements (cis-elements). The quantity of cis-elements was determined using the protein/DNA binding array membrane on which elements that would hybridize with any cis-elements present were pre-spotted. Cd treatment affected the binding of several transcription factors to cis-elements compared with the control group (Fig. 1c). Overall, Cd increased the binding activities of 20 transcription factors by more than 2-fold (Table 1). Our results showed that binding activities of both p53 and MTF-1 transcription factors were increased in HK-2 cells in response to Cd treatment. We previously demonstrated that p53 protein level was dramatically increased by Cd treatment in vivo and in vitro 14,15 . Moreover, another study demonstrated that the expression of apoptosis genes regulated by p53 is increased by Cd treatment in HK-2 cells 14 . A previous study showed that MTF-1 is induced to bind the MRE by Cd [23][24][25] . Our results are consistent with these findings. Cd also decreased the binding activities of 28 transcription factors, including PPAR (peroxisome proliferator activated receptor alpha) and Sp-1, by less than 0.5-fold (Table 2). Consistent with our results, several previous studies reported that PPAR and Sp-1 activities were decreased by Cd 28,29 . These studies help validate our findings and indicate our results may provide valuable information about the transcription factors involved in Cd toxicity or protection against Cd toxicity.

Identification of ARNT as a target transcription factor in Cd toxicity.
Our previous studies demonstrated that the suppression of gene expression is associated with Cd-induced renal toxicity 14,15,30,31 . These results imply that interruption in the regulation of gene expression is a key event in Cd toxicity. Therefore, we searched for a transcription factor that is involved in Cd toxicity, focusing on the transcription factors whose activities were decreased by Cd. In order to find the target transcription factor involved in Cd toxicity, it was determined whether the inhibition of expression of a transcription factor might affect the cell viability. As a result, each knockdown of six transcription factors triggered cell toxicity in HK-2 cells (Supplementary Table 1). Especially, the knockdown of ARNT [aryl hydrocarbon receptor nuclear translocator; known as hypoxia-inducible factor (HIF)-1β] by siRNA (Fig. 2a) conferred significant cell toxicity in HK-2 cells (Supplementary Table 1; Fig. 2b). EMSA assay showed that Cd treatment reduced the binding activity of ARNT (Fig. 2c). Knockdown of ARNT increased Cd toxicity in HK-2 cells (Fig. 2d), suggesting that decrease in the transcription activity of ARNT may strengthen Cd toxicity. In addition, although Cd did not affect the mRNA levels of ARNT (Fig. 2e), ARNT protein levels were decreased by Cd treatment in HK-2 cells (Fig. 2f). Together, this suggests that the ARNT transcription factor is a target in Cd-induced renal toxicity. BIRC3 as a responsible factor regulated by ARNT in Cd toxicity. We next examined which downstream factor of ARNT may be involved in Cd toxicity. Because ARNT binding activity was decreased by Cd and the knockdown of ARNT by siRNA decreased cell viability, we performed DNA microarray analysis to determine the gene expressions that were decreased in ARNT knockdown HK-2 cells. The results showed that the expressions of 27 genes were decreased by less than 0.5 fold in ARNT knockdown cells (Table 3). Interestingly, real-time RT-PCR showed that the gene expression of BIRC3 [baculoviral IAP (inhibitor of apoptosis protein) repeat containing 3; known as cIAP2], an apoptosis inhibitor, was decreased by both ARNT siRNA as well as Cd treatment (Fig. 3a,b). Moreover, the protein level of BIRC3 was drastically decreased by Cd treatment in HK-cells (Fig. 3c). To investigate whether the Cd-induced decrease in BIRC3 gene expression was mediated via ARNT, BIRC3 mRNA levels were examined in ARNT knockdown HK-2 cells treated with Cd (Fig. 3d). BIRC3 mRNA levels were decreased by Cd treatment in both control and ARNT knockdown cells. However, the significant decrease in BIRC3 gene expression by ARNT knockdown without Cd was vanished upon Cd treatment. To determine the significance, we performed statistical analysis (Supplementary Table 2). Even with the F(0.95) of siRNA*Cd as 3.89, the F value of 6.92 is within the rejection region. Moreover, the P value of siRNA*Cd is below 0.05. Therefore, the siRNA effect and Cd treatment effect is significantly dependent. These results suggest that Cd treatment reduces cellular BIRC3 level through the suppression of ARNT transcription activity. We next examined the impact of BIRC3 knockdown on cell viability using siRNA transfection. The mRNA and protein levels of BIRC3 were decreased by siRNA in a dose-dependent manner (Fig. 4a,b). Moreover, BIRC3 siRNA transfection significantly decreased HK-2 cell viability compared to control siRNA transfection cells (Fig. 4c). BIRC3 is a member of the BIRC family that is also known as the IAP family [32][33][34] . Eight family members have been identified in human, including BIRC1/NAIP, BIRC2/cIAP1, BIRC3/cIAP2, BIRC4/XIAP, BIRC5/ Survivin, BIRC6/Apollon, BIRC7/ML-IAP and BIRC8/ILP [32][33][34] . To determine whether BIRC3 is specifically involved in Cd renal toxicity, we next examined the effect of Cd treatment on the mRNA levels of all BIRC family members (Fig. 5). Although the mRNA level of BIRC4 was increased by Cd treatment, those of the other BIRC family members were unaffected by Cd treatment in HK-2 cells. These results suggest that specific reduction of BIRC3 levels by Cd-induced suppression of ARNT activity is involved in Cd renal toxicity.  Table 2. Transcription factors with decreased binding activities in response to Cd treatment. HK-2 cells were treated with 40 µM Cd in serum-free culture medium for 3 h. The cells were separated into nuclear and postnuclear fractions. The protein/DNA binding array was performed using the Combo Protein/DNA Array. Spot density was evaluated using ImageQuantTL software. Transcription factors whose activities were decreased 0.5fold or less are listed.
Scientific RepoRts | 7: 17287 | DOI:10.1038/s41598-017-17494-9 Cd-induced apoptosis through the suppression of BIRC3 expression. Previous studies showed that Cd induces apoptosis in proximal tubular cells 8,14,15 , and that BIRC3 inhibits apoptosis through the activity of caspases 35 . Therefore, we next examined whether BIRC3 is involved in Cd-induced apoptosis. Consistent with published studies, Cd treatment increased the level of cleaved caspase-3, the active form of casapse-3, in HK-2 cells (Fig. 6a). Moreover, knockdown of BIRC3 also increased the level of cleaved caspase-3 in HK-2 cells (Fig. 6b). These results suggest that knockdown of BIRC3 may induce apoptosis. Therefore, we performed TUNEL assays and found that knockdown of BIRC3 also induced apoptosis in HK-2 cells (Fig. 6c). Furthermore, knockdown of BIRC3 significantly increased Cd toxicity in HK-2 cells (Fig. 6d). Together, this demonstrates that a decrease in BIRC3 is involved in Cd-induced apoptosis in HK-2 cells.

Gene expression of BIRC3 and the activation of caspase-3 in several cultured cells. In addition
to Cd, other various metal(loid) toxicants also induce apoptosis in cultured cells. Methylmercury and inorganic mercury induce apoptosis in neuroblastoma and kidney cells, respectively [36][37][38] . The arsenic compound also causes apoptosis in hepatic cells 39,40 . To determine the specificity of BIRC3 function, we next examined the changes in BIRC3 gene expression and cleaved caspase-3 levels in several cell lines treated with metal(loid)s. Under the condition that methylmercury decreased the viability of human neuroblastoma (IMR-32 cells) by 20%, methylmercury increased cleaved caspase-3 levels; on the other hand, mRNA level of BIRC3 was not changed by methylmercury treatment (Fig. 7a-c). In HK-2 cells, methylmercury decreased cell viability by 10% as well as increased cleaved caspase-3 levels, but had no effect on BIRC3 mRNA levels ( Fig. 7d-f). Inorganic mercury decreased BIRC3 mRNA levels in HK-2 cells even when the viability was almost the same as the control group; on the other hand, although inorganic mercury treatment reduced viability of HK-2 cells to 60% compared to control, cleaved caspase-3 levels were unchanged ( Fig. 7g-i). In mouse hepatic cells (AML-12 cells), arsenic decreased the cell viability by 30%, with a significant increase in the mRNA level of Birc3 (Fig. 7j,l). Western blot analysis showed the protein levels of caspase-3 and cleaved caspase-3 were slightly increased by arsenic treatment (Fig. 7k). Although 6 h of Cd treatment (10 to 30 µM) decreased AML-12 cell viability to approximately 30%, only 10 µM Cd treatment decreased Birc3 mRNA levels (Fig. 7m,o). No cleaved caspase-3 levels were detected in AML-12 cells treated with Cd (Fig. 7n). As the western blot analysis did not clearly show the band of cleaved caspase-3 protein, we confirmed the analysis using staurosporin, an apoptosis inducer. Staurosporin drastically increased the protein level of cleaved caspase-3 in AML-12 cells (Supplementary Fig. 1). These data suggest that the activation of caspase-3 by suppression of BIRC3 gene expression by Cd treatment may mainly occur in proximal tubular cells.

Discussion
Here we have found that Cd induces apoptosis through the quantitative alleviation of apoptosis inhibitor, BIRC3, through the suppression of its transcription in human proximal tubular cells. Recent studies have reported that chronic Cd exposure can induce apoptosis in renal cells 8,[41][42][43][44][45][46] , and Cd induces via an ER-mediated pathway and mitochondrial-mediated pathway. In porcine renal proximal tubular epithelial LLC-PK1 cells, Cd induces ER stress and causes the activation of the unfolded protein response (UPR)-dependent apoptotic pathway, such as the IRE1-XBP1-JNK pathway 45,46 . In rat proximal tubule WKPT-0293 Cl.2 cells and mouse renal mesangial cells, Cd stimulates the release of pro-apoptotic factors from mitochondria 41,44 . Cd induces the swelling of mitochondria and subsequent cytochrome c release 42,43 . Our recent studies demonstrated that Cd induces apoptosis through p53 overaccumulation in human and rat proximal tubular cells [13][14][15] . In p53-mediated apoptosis, Cd inhibits the degradation of p53 by suppression of the gene expression of the UBE2D family, which is an E2 family enzyme in the UPS [13][14][15] . In the present study, we suggest a new apoptotic pathway involved in Cd toxicity. To the best of our knowledge, the ARNT-BIRC3 pathway is the first elucidated mechanism involved in Cd-induced apoptosis in proximal tubular cells (Fig. 8).
Our findings demonstrate that Cd suppresses the binding activity of the ARNT transcription factor in regulating BIRC3 expression. Our previous reports revealed that transcription factors FOXF1 and YY1 are involved in  Table 3. Downregulated genes in HK-2 cells transfected with ARNT siRNA (<0.5-fold).
the pathway by which Cd decreases the expression of genes coding for UBE2D proteins 14,27 . Therefore, together these results suggest that Cd induces apoptosis through the inhibition of anti-apoptosis proteins as well as the promotion of pro-apoptosis proteins. This also suggests that the initiation pathway responsible for Cd-induced apoptosis is the inhibition of transcription activity. Many studies have examined Cd toxicity, however only a few studies have reported the transcript pathway involved in Cd toxicity. A recent study reported that phosphorylation of the transcription factor FOXO3 promotes cell survival upon Cd treatment 47 . The upregulation of the transcription factor Snail by the activation of Notch1 signaling is reported to be involved in the Cd-induced decrease in cell-cell adhesion 48 . In this study, we propose a novel transcription factor ARNT in driving HK-2 cells to apoptotic cell death. Our protein/DNA binding array analysis in this study revealed that the binding activities of 48 transcription factors were affected by Cd treatment. Our previous study determined that Cd decreased the activity of the FOXF1 transcription factor, which suppressed the downstream factor UBE2D4, leading to apoptosis through p53 overaccumulation 14 . Our results identified FOXF1 as one of the transcription factors whose activities were decreased by Cd treatment (Table 2). In Supplementary table 1, the list of transcription factors whose gene knockdown affects cell viability is shown. In addition to ARNT and FOXF1, such transcription factors as GATAs and MEF2A may play essential roles in the pathway of Cd toxicity. Moreover, an increase in DNA binding activity of the apoptotic-related transcription factor p53, which was accumulated in proximal tubular cells by Cd, was detected in our protein/DNA binding array upon Cd treatment (Table 1). Thus, the results from our protein/ DNA binding array can propose useful and valid information to help elucidate transcription factors involved in Cd toxicity.
ARNT is a member of the basic helix-loop-helix/Per-ARNT-Sim family. Previous studies showed that ARNT heterodimerizes with the aryl hydrocarbon receptor (AhR) or oxygen sensitive alpha subunit (HIF-1α or HIF-2α) [49][50][51] . AhR is a ligand-binding protein that functions as the response factor to environmental pollutant exposure, including ligands such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin 52,53 . The non-ligand bound AhR binds instead to two heat shock proteins (XAP2 and HSP90) and the cochaperone p23 in the cytosol 52 . Ligand binding disrupts the complex and causes AhR to translocate into the nucleus. After heterodimerization with ARNT, AhR/ARNT binds to xenobiotic response elements (XREs) or dioxin responsive elements (DREs) in the promoter sequences of target genes 52 . In normoxia, HIF-1α and HIF-2α are hydroxylated and rapidly degraded by the UPS 50,54 . In hypoxia, HIF-1α and HIF-2α are stabilized and bind to ARNT to regulate the expression of downstream target genes 50,54 . HIF-mediated pathways are essential cellular responses to hypoxia such as angiogenesis, erythropoiesis, cell growth, and cell differentiation [54][55][56][57] . Nonetheless, previous studies suggested that independent of its role in AhR and HIF signaling, ARNT has new regulatory functions in the expression of cyclooxygenase-2, 12(S)-lipoxygenase, and p21 genes under normoxia conditions [58][59][60] . In addition, ARNT is associated with the proliferation and survival of tumor cell lines by regulating cellular processes 49,58 . These observations, including our findings, indicate that ARNT activity is not only induced by such signals as AhR and HIF pathway but is also suppressed by signals apart from the AhR or HIF pathway. In this study, HIF-1 activity was decreased by Cd treatment (Table 2); therefore, Cd-mediated suppression of ARNT activity may involve the AhR and HIF pathway. Further studies are required to elucidate whether the AhR or HIF pathway may be associated with Cd-suppressed ARNT activity.
Apoptosis is a highly programmed pathway of cell death that is typically achieved through the activation of caspases 35 . During death receptor-or mitochondrial-mediated apoptosis, initiator caspases, such as caspase-8/9 and -10, are recruited via binding proteins FADD, cytochrome c and Apaf-1 61,62 . After recruitment of initiator caspases, effector caspases such as caspase-3 and -7 are activated by initiator caspases following cleavage into large and small subunits 63,64 . Cells have evolved important mechanisms to regulate caspase activity, for example, using the BIRC family. BIRC family members possess one or more baculorvirus IAP repeat (BIR) domains that selectively inhibits the activity of caspase-9, -3, or -7 [32][33][34] . BIRC4, also known as XIAP (X-linked IAP), is the most characterized member of the family 35 . BIRC4 blocks apoptosis by inhibiting caspases, using the interaction its BIR2 and BIR3 regions with active-site pocket of caspases 65 . Based on the mechanism of BIRC4, most BIRC family members were suggested to neutralize caspase activities in the same manner. However, recent studies have reported that BIRC family members are functionally non-equivalent and regulate caspase activities via distinct mechanisms [66][67][68] . BIRC2, BIRC3 and BIRC4 contain a C-terminal RING zinc finger domain with E3 ubiquitin ligase activity that mediates proteasomal degradation of cellular targets as well as themselves 69 . BIRC2 was demonstrated to interact with caspase-7 independently on the active site pocket 66 . BIRC2 ubiquitinates active caspase-3 and -7 and mediates their proteasomal degradation, thereby suppressing apoptosis 67 . In this study, the decrease in BIRC3 protein level increased the level of the active form of caspase-3. This suggests that BIRC3 may be directly involved in degradation of the active form of caspase-3 rather than interrupting the active site of caspase-3 in HK-2 cells. Further studies are required for elucidating the precise mechanism underlying BIRC3-mediated caspase-3 deactivation upon Cd treatment.
Our most striking observation in this study is our novel finding, to the best of our knowledge, that the ARNT-BIRC3 pathway is involved in Cd-induced apoptosis in proximal tubular cells. The regulation of BIRC3 gene expression has been previously unknown; however, our study suggests that ARNT may be a key transcription factor in BIRC3 regulation. Finally, we provide valuable information about the critical transcription factors for elucidating the toxic and defense mechanism pathways in response to Cd.  DMEM supplemented with 10% FBS, 25 U/mL penicillin, 25 µg/mL streptomycin, and 1% MEM Non-essential Amino Acid Solution (Sigma-Aldrich) at 37 °C in a humidified incubator containing 5% CO 2 .
HK-2 cells and AML-12 cells were grown in plates at a density of 250 cells/mm 2 and cultured for 48 h. IMR-32 cells were grown in plates at a density of 500 cells/mm 2 and cultured for 48 h. The culture medium was discarded and the cells were treated with Cd (CdCl 2 ; Wako Pure Chemical Industries, Osaka, Japan), methylmercury (CH 3 HgCl; GL Sciences Inc., Tokyo, Japan), inorganic mercury (HgCl 2 ; Wako Pure Chemical Industries), or arsenic (NaAsO 2 ; Wako Pure Chemical Industries) in serum-free culture medium for various times.   Nuclear extraction. Nuclei were extracted with the Nuclear Extraction Kit (Panomics; Affymetrix, Santa Clara, CA, USA). After Cd treatment, HK-2 cells were washed twice with ice-cold PBS and lysed on ice for 10 min in extraction buffer A including a protease inhibitor, phosphatase inhibitor, and dithiothreitol (DTT). The cells were harvested from the assay plates by scraping and pipetting up and down several times to disrupt the cell clumps. Nuclei were collected by centrifugation at 14,000 × g for 3 min at 4 °C. The pellet was resuspended in extraction buffer B including a protease inhibitor, phosphatase inhibitor, and DTT and incubated at 4 °C for 1 h. The mixture was then centrifuged at 14,000 × g for 5 min at 4 °C, and the supernatant was collected. Protein concentration was measured using the BCA protein assay kit.
Protein/DNA binding array. HK-2 cells were grown in 60-mm dishes at a density of 250 cells/mm 2 and cultured for 48 h. Culture medium was discarded and the cells were treated with 40 µM Cd in serum-free culture medium for 3 h. HK-2 cells were separated into nuclear and post-nuclear fractions. The protein/DNA binding array was performed using the Combo Protein/DNA Array (Affymetrix) 27 . In brief, 20 μg of nuclear extracts were mixed with a biotin-labeled probe mix, and the mixture was incubated at 15 °C for 30 min. The protein-bound probes in the mixture were isolated from the non-bound probes using a spin column. The protein-bound probes were eluted with column elution buffer and denatured at 95 °C for 3 min. The eluted probes were then added to the hybridization buffer and hybridized to the array membrane spotted with 345 consensus sequences complementary to the probes at 42 °C overnight. The membrane was washed twice in 2 × saline sodium citrate (SSC)/0.5% sodium dodecyl sulfate (SDS) at 42 °C for 20 min and then twice in 0.1 × SSC/0.5% SDS at 42 °C for 20 min. The membrane was then blocked with 1 × blocking buffer. The biotin-labeled probes were detected with streptavidin-horseradish peroxidase diluted 1:10000. The image was acquired using an LAS-4000 device. Spot density was evaluated using ImageQuantTL software (GE Healthcare).
Gel shift assay. The gel shift assay was performed using the EMSA kit purchased from Panomics (Affymetrix). HK-2 cells were grown in 60-mm dishes at a density of 250 cells/mm 2 and cultured for 48 h. After treatment, HK-2 cells were separated into nuclear and post-nuclear fractions. Nuclear protein (3 µg) was incubated with 10 ng DNA probe (biotin-labeled binding sequence to transcription factor) and 1 µg poly d(I-C) with binding buffer for 30 min at 15 °C in a thermal cycler (Takara Bio, Shiga, Japan). For the competition assay, 1,320 ng cold DNA probe was added. The protein-bound probe was electrophoresed on a 5.0% (w/v) TBE (Tris borate EDTA)-polyacrylamide gel in 0.5 × TBE buffer at 4 °C and then transferred to a Biodyne ® B nylon membrane (Pall Corporation, Port Washington, NY, USA) in 0.5 × TBE buffer. The membrane was fixed by UV crosslinking (CL-1000 Ultraviolet Crosslinker; UVP, Upland, CA, USA) with 120 mJ/cm 2 . The membrane was blocked and probed with Streptavidin-HRP. The chemiluminescence images were taken using a LAS-3000 device. siRNA transfection. Silencer Select Pre-designed siRNAs were purchased from Ambion (Grand Island, NY, USA) as follows: s1613 and s1615 (Silencer ® Select Pre-designed siRNA) for human ARNT; and s1451, s1452 and s1453 (Silencer ® Select Pre-designed siRNA) for human BIRC3. Control siRNA (Silencer ® Select Negative Control #1 siRNA) was also purchased from Ambion. siRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen). After the siRNA mixture was incubated for 15 min with Lipofectamine RNAiMAX and $ Significantly different from the control group of 6 h treated group, P < 0.05. # Significantly different from the control group of 24 h treated group, P < 0.05. The absence of an error bar indicates that the S.D. was within the area of the symbol. (b, e, h, k, n) Whole cell lysates were used for western blot analysis and probed with caspase-3 or cleaved caspase-3 antibody. GAPDH was probed as a loading control. The blots were run under the same experimental conditions and cropped from same membrane. Uncropped images are provided in Supplementary Fig. 3. (c, f, i, l, o) mRNA level of BIRC3 or Birc3 was examined using real-time RT-PCR. mRNA levels were normalized with GAPDH or β-actin. Values are the means ± S.D. (n = 3). *P < 0.05 vs. control.