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. 2019 Jan 1;15(2):369–385. doi: 10.7150/ijbs.28422

Figure 5.

Figure 5

ING4 represses HCC via upregulating expression and transcriptional activity of FOXO3a. A, B. Western blot analysis of FOXO3a and its targets. The total lysates of MHCC97H-ING4 vs MHCC97H-mock and MHCC97L-shING4 vs MHCC97L-shcontrol HCC cells were immunoblotted with anti-FOXO3a, anti-p27, anti-Cyclin D1, anti-Bim, anti-Puma, anti-FasL, anti-TRAIL, anti-β-catenin or anti-β-actin (a loading control) antibody. The nuclear lysates derived from above cells were immunoblotted with anti-FOXO3a, anti-β-catenin or Histone H3 (a loading control) antibody. The representative pictures of Western blot were shown (A). The total expression level of FOXO3a was normalized to β-actin (FOXO3a/β-actin); the nuclear expression level of FOXO3a was normalized to Histone H3 (FOXO3a/Histone H3), and then expressed as a ratio or fold of respective control, with 1 being the value for MHCC97H-mock or MHCC97L-shcontrol control (B). MHCC97H: *, p<0.05 compared with MHCC97H-mock; MHCC97L: *, p<0.05 compared with MHCC97L-shcontrol, Student t test, n=6 replicates per sample. C. Real-time qRT-PCR analysis of FOXO3a. The mRNA level of FOXO3a was normalized to β-actin and calculated by a 2-∆∆CT method, with 1 being the value for MHCC97H-mock or MHCC97L-shcontrol control. MHCC97H: *, p<0.05 compared with MHCC97H-mock; MHCC97L: *, p<0.05 compared with MHCC97L-shcontrol, Student t test, n=6 replicates per sample. D. Luciferase reporter analysis of transcriptional activity of FOXO3a and β-catenin. The luciferase activity was expressed as a ratio or fold of MHCC97H-mock or MHCC97L-shcontrol control, with 1 being the value for controls. MHCC97H FOXO3a or β-catenin: *, p<0.05 compared with MHCC97H-mock; MHCC97L FOXO3a or β-catenin: *, p<0.05 compared with MHCC97L-shcontrol, Student t test, n=6 replicates per sample. E. Western blot analysis after FOXO3a siRNA knockdown. The total or nuclear lysates derived from the siFOXO3a- or sicontrol-transfected MHCC97H-ING4 cells and the untransfected MHCC97H-ING4 cells were immunoblotted with a panel of antibodies as described above. The representative pictures of Western blot were shown. F. CCK-8 assay after FOXO3a siRNA knockdown. *, p<0.05 compared with MHCC97H-ING4 and sicontrol-transfected MHCC97H-ING4 at day 2, 3 and 4, respectively, two-way repeated measures ANOVA, n=6 replicates per condition. G. Apoptosis analysis after FOXO3a siRNA knockdown. *, p<0.05 compared with MHCC97H-ING4 and sicontrol-transfected MHCC97H-ING4, one-way repeated measures ANOVA, n=6 replicates per condition. H. Transwell migration assay after FOXO3a siRNA knockdown. *, p<0.05 compared with MHCC97H-ING4 and sicontrol-transfected MHCC97H-ING4, one-way repeated measures ANOVA, n=6 replicates per condition, n=5 observations per replicate. I. Transwell invasion assay after FOXO3a siRNA knockdown. *, p<0.05 compared with MHCC97H-ING4 and sicontrol-transfected MHCC97H-ING4, one-way repeated measures ANOVA, n=6 replicates per condition, n=5 observations per replicate. J, K. Subcutaneous xenograft mouse model after FOXO3a siRNA knockdown. The tumor volume (J) was measured after implantation of tumor cells. *, p<0.05 compared with MHCC97H-ING4 and sicontrol-transfected MHCC97H-ING4 at day 2, 3 and 4, respectively, two-way repeated measures ANOVA, n=6 replicates per condition. The xenografted tumors were removed 4 weeks after tumor cell's implantation and tumor weight (K) was then measured. *, p<0.05 compared with MHCC97H-ING4 and sicontrol-transfected MHCC97H-ING4, one-way repeated measures ANOVA, n=6 replicates per condition. L. In vivo lung metastasis assay after FOXO3a siRNA knockdown. *, p<0.05 compared with MHCC97H-ING4 and sicontrol-transfected MHCC97H-ING4, one-way repeated measures ANOVA, n=6 replicates per condition, n=5 sections per sample, n=5 observations per section. Data shown were representative of three independent experiments.