Additional file 3 of Downregulation of the ubiquitin ligase KBTBD8 prevented epithelial ovarian cancer progression

Additional file 3: Figure 1. Results in HO8910 showed that human KBTBD8 was important for the proliferation of ovarian epithelial cancer cells. A and B Blot and quantification showed that KBTBD8 protein level was about tenfold higher in HO8910 ovarian cancer cells than in normal cell line moody (KBTBD8 protein level, Moody vs. HO8910, 0.01478 vs. 0.1490). C and D Blot and quantification showed that KBTBD8 protein was significantly reduced by specific shRNA. E and F The number of colonies formed after 7 days was significantly decreased in KBTBD8-knockdown (K8-KD) compared with the numbers in control cells (colony numbers, CTR vs. K8-KD, 135 vs. 49). G–I Cell cycle analysis by FACS showed that significantly fewer cells were at S stage (proportion of cells at S phase, CTR vs. K8-KD, 45.88% vs. 21.24%) and considerably more cells were at the G1 stage (percentage of cells at the G1 phase, CTR vs. K8-KD, 48.37% vs. 69.90%) in KBTBD8-knockdown HO8910 cells than in control cells. J CCK8 assay also showed that there were significantly less proliferating cells in the KBTBD8-knockdown HO8910 cells than in control cells (CCK8, CTR vs. K8-KD, 0.8500 vs. 0.6259). A.U., arbitrary unit. *Indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. Figure 2: Results in HO8910 cells showed that human KBTBD8 was important for the migration of EOC cells. A and B Would healing assays showed that the closure ratio of the scratched area was slower in the KBTBD8-knockdown HO8910 cells than in the control cells. Representative images at 0 h, 24 h, 48 h, and 72 h were shown. Percentage of initial area uncovered by migrated cells at 24 h, CTR vs. K8-KD, 56.03% vs. 86.67%; at 48 h, 20.34% vs. 66.68%; at 72 h, 8.719% vs. 55.31%. C and D 3-dimensional migration assay showed that the migrated cells were significantly less in the KBTBD8-knockdown than in the control cells (Number of migrated cells per field, CTR vs. K8-KD, 216 vs. 22). Scale bar, 50 μm. ****Indicates p < 0.0001. Figure 3: Results in HO8910 showed that human KBTBD8 was important for the general health state of ovarian epithelial cancer cells. A and B KBTBD8 knockdown significantly increased ROS level by eightfold in HO8910 ovarian cancer cells (ROS level, CTR vs. K8-KD, 1.508 vs. 12.26). C and D JC-1 staining assay showed that the mitochondria membrane potential significantly decreased by tenfold after the KBTBD8 knockdown (JC1 level, CTR vs. K8-KD, 1.390 vs. 0.1330). Aggregate in green, monomer in red. E and F Annexin V staining assay showed that KBTBD8 knockdown significantly increased the apoptosis level (Annexin V level, CTR vs. K8-KD, 1.857 vs. 5.738). Annexin V signal in green, PI in red. Scale bar, 50 μm. ***Indicates p < 0.001, **** indicates p < 0.0001. Figure 4: KBTBD8 knockdown decreased the overall ubiquitination level in A2780 EOC cells. A-C related to Fig. 6d and e, three repeats all showed that KBTBD8 knockdown decreased the overall ubiquitination level in A2780 EOC cells. Figure 5: KBTBD8 knockdown suppressed tumor formation in vivo in HO8910 cell xenograft model. A–C In vivo tumor formation assay in HO8910 cell xenograft model. Both tumor volume (tumor volume at 21d, CTR vs. K8-KD, 490.4 mm3 vs. 138.2 mm3) and weight (tumor weight at 21d, CTR vs. K8-KD, 0.5777 vs. 0.1451) were significantly reduced in the KBTBD8-knockdown HO8910 cell xenograft group. D and E Western blot and quantification showed that the KBTBD8 protein level was still significantly lower (KBTBD8 level, CTR vs. K8-KD, 0.1385 vs. 0.02099) in the KBTBD8-knockdown group at the examination time point. F Immunohistochemistry of tumor tissues verified that KBTBD8 signal was still significantly lower in the KBTBD8-knockdown group at the examination time point (Three weeks after the xenograft). G and H Immunofluorescence also showed that KBTBD8 signal was still significantly lower (KBTBD8 signal, CTR vs. K8-KD, 1.033 vs. 0.01459) in the KBTBD8-knockdown group at the examination time. KBTBD8 in red, DNA in blue. I and J Tunel assay showed that apoptosis signal significantly increased in KBTBD8-knockdown tumor than in control (Tunel level, CTR vs. K8-KD, 1.117 vs. 1.539). Tunel signal in green, DNA in blue. Scale bar, 50 μm. **Indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.