XIAP promoter predicted GLI-binding sites exhibit sequence-specific binding to nuclear proteins.

<p><i>A,</i> Putative GLI binding sites in the XIAP promoter region. Nucleotide positions were counted from the transcription start site (TSS). Potential binding sites were observed as indicated by arrows on the schematic (I and II). <i>B,</i> Nuclear protein extracts were isolated from KMCH cells. EMSA was performed using Cy5.5-labeled double-stranded oligonucleotides containing the putative GLI binding sequences within the human XIAP promoter, and competition experiments with 200-fold molar excess cold oligonucleotides as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018330#s2" target="_blank">MATERIALS AND METHODS</a>”. <i>C</i>, Total cellular protein was isolated 48 hours after transient transfection with the indicated siRNA against GLI family members. XIAP protein expression was determined by immunoblotting and the signal was quantified by densitometry as a ratio to actin expression. <i>D,</i> Chromatin immunoprecipitation using antiserum to GLI1, GLI2, GLI3, or a control IgG was performed followed by PCR using primers flanking site I (341 bp) within the <i>XIAP</i> promoter region, as indicated. As a positive control, ChIP was performed using primers flanking the <i>Bcl-2</i> promoter GLI-binding site (GLI1 and GLI2), or primers flanking the GLI3 binding site within the <i>GLI1</i> promoter.</p

XIAP promoter predicted GLI-binding sites exhibit sequence-specific binding to nuclear proteins.

<p><i>A,</i> Putative GLI binding sites in the XIAP promoter region. Nucleotide positions were counted from the transcription start site (TSS). Potential binding sites were observed as indicated by arrows on the schematic (I and II). <i>B,</i> Nuclear protein extracts were isolated from KMCH cells. EMSA was performed using Cy5.5-labeled double-stranded oligonucleotides containing the putative GLI binding sequences within the human XIAP promoter, and competition experiments with 200-fold molar excess cold oligonucleotides as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018330#s2" target="_blank">MATERIALS AND METHODS</a>”. <i>C</i>, Total cellular protein was isolated 48 hours after transient transfection with the indicated siRNA against GLI family members. XIAP protein expression was determined by immunoblotting and the signal was quantified by densitometry as a ratio to actin expression. <i>D,</i> Chromatin immunoprecipitation using antiserum to GLI1, GLI2, GLI3, or a control IgG was performed followed by PCR using primers flanking site I (341 bp) within the <i>XIAP</i> promoter region, as indicated. As a positive control, ChIP was performed using primers flanking the <i>Bcl-2</i> promoter GLI-binding site (GLI1 and GLI2), or primers flanking the GLI3 binding site within the <i>GLI1</i> promoter.</p

Cyclopamine sensitizes cells to TRAIL-induced cell death independent of Bax and Bak.

<p><i>A,</i> Whole cell lysates from KMCH cells transiently transfected with specific siRNA targeting Bax and/or Bak or scrambled siRNA were obtained 48 hours after transfection and analyzed by immunoblot using anti-Bax or anti-Bak antisera. Actin was used as a loading control. <i>B</i>, KMCH cells transiently transfected with Bax- and Bak-siRNA or scrambled siRNA were pretreated (24 hours) with medium or cyclopamine (5 µM) as indicated, then treated with Fas agonistic antibody CH-11 (100 µg/mL) for 5 hours. Cells with apoptotic nuclear morphology were counted. Mean +/− SEM; ***p<0.001. <i>C,</i> KMCH cells were treated as in panel <i>B</i> and caspase-3/7 activity was measured after 5 hours. Mean +/− SEM; ***p<0.001 <i>D</i>, KMCH cells transiently transfected with Bax- and Bak-siRNA or scrambled siRNA were pretreated with vehicle or cyclopamine (5 µM) for 24 hours, and then treated with TRAIL (5 ng/mL) for 5 hours. Cells with apoptotic nuclear morphology were counted. Mean ± SEM, #p<0.05 and ***p<0.001 compared to cells transfected with scrambled siRNA and treated with TRAIL. <i>E</i>, KMCH cells transiently transfected with indicated siRNA and treated as in panel <i>D</i>, were assayed after 5 hours for caspase-3/7 activity. Mean ± SEM; ***p<0.001.</p

Cyclopamine sensitizes cells to TRAIL-induced cell death despite knockdown of Bid or Bim.

<p><i>A,</i> Immunoblot analysis was performed on whole cell lysates obtained from KMCH cells stably transfected with the specific shRNA targeting <i>Bid</i> or <i>Bim,</i> using indicated antisera. Actin was used as a loading control. <i>B</i>, KMCH cells stably transfected with Bid- or Bim-shRNA and untransfected parental KMCH cells were treated with Fas agonistic antibody CH-11 (100 µg/mL) for 5 hours followed by DAPI staining. Cells with apoptotic morphology were counted. Mean ± SEM, ***p<0.001 compared to parental cells treated with CH-11. <i>C,</i> In parallel, caspase-3/7 activity was measured in cells treated as in panel <i>B</i>. Mean ± SEM, ***p<0.001. <i>D</i>, KMCH cells stably transfected with Bid- or Bim-shRNA were pretreated with vehicle or cyclopamine (5 µM) for 24 hours, and then treated with TRAIL (5 ng/mL) for 5 hours followed by DAPI staining. Cells with apoptotic morphology were counted. Mean ± SEM, ***p<0.001 compared to cells treated with TRAIL alone. <i>E</i>, KMCH stable cell lines were treated as in panel <i>D</i>, and after 5 hours caspase-3/7 activity was measured. Mean ± SEM, ***p<0.001 compared to cells treated with TRAIL alone. <i>F</i>, Whole cell lysates from KMCH, HuCCT-1, and Mz-ChA-1 cells treated with vehicle or cyclopamine (5 µM) for 24 hours were analyzed by immunoblot using anti-Bcl-2, Mcl-1, or Bcl-x antisera. Actin was used as a loading control. <i>G</i>, Whole cell lysates were obtained from shBid-, shBim- and untransfected parental KMCH cells treated with vehicle or cyclopamine (5 µM) for 24 hours and were analyzed by immunoblot using anti-Bcl-2, Mcl-1, or Bcl-x antisera. Actin was used as a loading control.</p

Knockdown of XIAP sensitizes human cholangiocarcinoma cells to TRAIL cytotoxicity.

<p>KMCH cells were transfected with scrambled siRNA or specific siRNA for <i>cIAP-1</i> or <i>XIAP</i>. <i>A</i>, Whole cell lysates were prepared for immunoblotting from KMCH cells 48 hours after siRNA transfection. <i>B</i>, Forty-eight hours after transfection as in panel <i>A</i>, cells were treated with human recombinant TRAIL (5 ng/mL) or medium for 5 hours. Cells were then stained with DAPI and cells with apoptotic nuclear morphology were counted and expressed as a percentage of total nuclei. <i>C,</i> In parallel, cells were transfected and treated with TRAIL as in panel <i>B</i>, and after 5 hours caspase-3/7 activity was measured. Mean ± SEM, *p<0.05; **p<0.01; ***p<0.001.</p

XIAP knockdown sensitizes cells to TRAIL-induced cell death despite Bid inhibition.

<p><i>A,</i> KMCH cells were incubated for 24 hours in medium or cyclopamine (5 µM) followed by treatment with either vehicle (DMSO, final concentration 0.1% v/v) or the Bid inhibitor, BI-6C9 (10 µM) for 1 hour. Cells were subsequently treated with Fas agonistic antibody CH-11 (100 µg/mL) for an additional 5 hours (where appropriate, BI-6C9 remained in the medium during CH-11 treatment). Cells with apoptotic nuclear morphology were quantified as referenced in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018330#s2" target="_blank">Materials and Methods</a>. Mean +/− SEM; ***p<0.001; n.s. indicates p>0.10 by ANOVA with Bonferroni correction. <i>B,</i> KMCH cells were incubated overnight with or without cyclopamine followed by BI-6C9 as in panel <i>A</i>, except that apoptosis was induced by TRAIL (5 ng/mL) for 5 hours (where appropriate, BI-6C9 remained in the medium during TRAIL treatment) and apoptotic nuclear morphology was quantified. Mean +/− SEM; ***p<0.001; n.s. indicates p>0.10 by ANOVA with Bonferroni correction. <i>C,</i> KMCH cells transiently transfected with XIAP siRNA or scrambled siRNA (48 hours) were pretreated with BI-6C9 (10 µM) or DMSO vehicle (final concentration 0.1% v/v) for 1 hour. Next, the cells were treated with TRAIL (5 ng/mL) for 5 hours (where appropriate, BI6C9 remained in the medium during TRAIL treatment). Cells with apoptotic nuclear morphology were quantified. Mean ± SEM, *p<0.05; n.s. indicates p>0.10 by ANOVA with Bonferroni correction.</p

Sequences for real-time PCR primers.

<p>Sequences for real-time PCR primers.</p

Smoothened inhibition down-regulates both cIAP-1 and XIAP expression.

<p><i>A,</i> Cell lysates from KMCH cells treated with cyclopamine (5 µM) for the indicated times were immunoblotted for cIAP-1, cIAP-2 and XIAP. Actin was used as a loading control. <i>B,</i> KMCH cells were transfected with a tetracycline-inducible shRNA expression vector targeting Smoothened (shSMO) or a scrambled shRNA vector; shRNA expression was induced for 48 hours by treatment with tetracycline (1 mg/mL) followed by immunoblotting for cIAP-1, cIAP-2 and XIAP using whole cell lysates. <i>C</i>, Cells were treated with vehicle, cyclopamine (5 µM) for 24 hours, or transfected with shSMO as in panel <i>B,</i> then analyzed by real-time RT-PCR for <i>cIAP-1</i>, <i>cIAP-2</i> and <i>XIAP</i> using total RNA. <i>D</i>, Total RNA from tissue samples of either cholangiocarcinoma (CCA) or adjacent benign liver was used to assess <i>XIAP, cIAP-1, and cIAP-2</i> mRNA expression, normalized to 18S and expressed as fold change from benign. Mean ± SEM, *p<0.05; **p<0.01; ***p<0.001 compared to vehicle.</p