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hep26705-sup-0001-supptableS1.tif4403KTable. SI Clinicopathological factors and Gankyrin expression in 85 CCA patients.
hep26705-sup-0002-supptableS2.tif1345KTable. SII Univariate and multivariate analyses of factors associated with overall survival in CCA patients. OS indicates overall survival; HR, hazard ratio; CI, confidence interval. P, Poor; W, Well; M, Moderate.
hep26705-sup-0003-suppfigS1.tif1113KFig. S1 All collected CCA samples were immunostained with a monoclonal anti-Gankyrin antibody. Representative Gankyrin-negative (N: no positive Gankyrin staining), Gankyrin-low (L: below the median value of the integrated optical density) and Gankyrin-high (H: above the median value of the integrated optical density) samples were shown.
hep26705-sup-0004-suppfigS2.tif2175KFig. S2 (A) Invasion assays for the indicated four CCA cell lines. (B) Growth curves of the indicated CCA cells were evaluated by the trypan blue dye exclusion method. All experiments were done three times and data were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
hep26705-sup-0005-suppfigS3.tif617KFig. S3 (A) The plasmid for the construction of lentivirus mediated Gankyrin shRNA. (B) High levels of GFP fluorescence in the Lenti-Negative control (NC) and Lenti-shRNA Gankrin-1 (KD-1) cells, while there is no GFP fluorescence in the Control (CON) cells. (C) Gene silencing by lentiviral delivery of Gankyrin shRNAs. In the study with different CCA cells, expression of Gankyrin was lower after blocking by shRNA-1 (KD1) than the other three sequences. CON, control group without any infection; NC, infected with negative lentivirus; KD-1, infected with Lenti-shRNA-1.
hep26705-sup-0006-suppfigS4.tif2622KFig. S4 (A) Wounds were introduced by scratching a monolayer of indicated CCA cells. Migration was monitored by light microscopy at 0h and 24h. The widths of the gaps from three experiments were measured, and the results are presented as a bar graph. ***P < 0.001. (B) Migration assays of the indicated CCA cell lines. Cells migrated to the bottom of the chamber were counted in three fields under ×20 magnification. Experiments were done three times and data were presented as mean ± SD. **, P < 0.01; ***, P < 0.001. CON, control group without any infection; NC, infected with negative lentivirus; KD-1, infected with Lenti-shRNA-1.
hep26705-sup-0007-suppfigS5.tif1353KFig. S5 (A) Densitometric analysis showing the relative p-Rb:Rb ratio in the CCA (CON, NC and KD-1) cells. **, P < 0.01; ***, P < 0.001. (B) Characterization of Rb or Gankyrin knockdown by transient transfection of siRNAs in CCA cell lines. After CCA cells were transfected with siRNAs against Rb and Gankyrin for 48h, the expression of Rb and Gankyrin was investigated by Western blot analysis. Lane 1: siRNA control; lane 2 siRNA Rb and Gankyrin.
hep26705-sup-0008-suppfigS6.tif1745KFig. S6 Gankyrin knockdown reduces E2F-1 binding to Cyclin A and Cyclin E promoters. (A) Results of CHIP with an anti-E2F-1 antibody in different CCA cell extracts followed by PCR for Cyclin A. (B) Results of CHIP with an anti-E2F-1 antibody in different CCA cell extracts followed by PCR for Cyclin E. Gel showing PCR bands for Input (Lane 1, positive control), IgG (Lane 2, negative control) and E2F-1 (Lane 3).
hep26705-sup-0009-suppfigS7.tif1072KFig. S7 Effects of Gankyrin knockdown on the apoptosis of CCA cells. CCA cells (CON, NC or KD-1) were subjected to cell apoptosis assays. The apoptosis rates were quantified and presented as mean ± SD. CON, control group without any infection; NC, infected with negative lentivirus; KD-1, infected with Lenti-shRNA Gankyrin-1.
hep26705-sup-0010-suppfigS8.tif808KFig. S8 Characterization of Gankyrin overexpression in different CCA cell lines. TFK-1 and HuH-28 cells were stably transfected with pCMV-Tag2-hGankyrin plasmid or pCMV-Tag2B empty vector. The resulting clones (CCA-Gankyrin) were screened for the greatest degree Gankyrin overexpression compared to the vector control-transfected cell line by Western blot
hep26705-sup-0011-suppfigS9.tif2684KFig. S9 (A) Expressions of p-ERK, ERK, p-Akt, Akt, p-JNK, JNK and actin was determined by Western blot in HuCC-T1 control and Gankyrin knockdown cells. (B) Expression of p-ERK, ERK, p-Akt, Akt, p-JNK, JNK and β-actin was determined by Western blot in TFK-1 control and Gankyrin overexpressed cells. (C) Densitometric analysis showing the relative p-STAT3:STAT3 ratio in the shRNA con and shRNA Gankyrin CCA (QBC939, HuH-29, TFK-1 and HuCC-T1) cells from Fig. 5A. (D) Densitometric analysis showing the relative p-STAT3:STAT3 ratio in the vector and Gankyrin ovexpressed CCA (QBC939 and TFK-1) cells from Fig. 5B. *, p<0.05 **, P < 0.01; ***, P < 0.001.
hep26705-sup-0012-suppfigS10.tif2602KFig. S10 (A) Characterization of STAT3 knockdown by transient transfection of siRNA against STAT3 in TFK-1-Con and TFK-1-Gankyrin stable cell lines. After cells were transfected with siRNA against STAT3 for 48h, the expression of STAT3 and p-STAT3 was investigated by Western blot analysis. (B) Soft agar and matrigel invasion assays were performed for the indicated cells following AG490 treatment. Experiments were done three times and data were presented as mean ± SD. ***, P < 0.001.
hep26705-sup-0013-suppfigS11.tif5074KFig. S11 (A) Correlation between Gankyrin expression and p-STAT3 level was examined in tumor tissues derived from 67 patients, r=0.4278, P= 0.0003. (B) Representative immunostaining for Gankyrin and p-STAT3 was shown for two patient samples (Case 12 and 34) with high or low Gankyrin respectively.
hep26705-sup-0014-suppfigS12.tif3706KFig. S12 (A) Densitometric analysis showing the relative p-Rb:Rb ratio and p-STAT3:STAT3 ratio in Fig. 6A-B. (B) Immunostaining of CD31 was performed, and the microvessel density (MVD) was calculated in xenograft tumors from each group. Results display the mean ± SD from triplicate experiments. ***, P < 0.001.
hep26705-sup-0015-suppfigS13.tif1745KFig. S13 (A) Densitometric analysis showing the relative p-Rb:Rb ratio of the HuCCT-1 cells in Fig. 7C. **, P < 0.01. (B) Characterization of CCA-IL-6-shRNA cell lines. TFK-1 and HuH-28 cells were stably transfected with human IL-6-shRNA plasmid. The clones (CCA-IL-6-shRNA) were screened for the greatest degree of IL-6 knockdown compared to the control-transfected cell line by real-time PCR. Data were presented as relative IL-6 expression (mean ± SD) after normalizing to β-actin levels. (*p<0.05).
hep26705-sup-0016-suppfigS14.tif1009KFig. S14 rIL-6 significantly increases the expression of Gankyrin in IL-6 shRNA CCA cells. shRNA IL-6 TFK-1 and shRNA IL-6 HuH-28 cells were treated with rIL-6 (10ng/ml) for indicated time points. Gankyrin expression was assessed by Western blot. Representative Gankyrin immunoblots were shown.
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