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. 2021 Nov 20;7:361. doi: 10.1038/s41420-021-00605-x

Fig. 3. FBW7 inhibits TNF-α-induced pancreatic beta-cell apoptosis and dysfunction through ubiquitin degradation of EZH2.

Fig. 3

A Western blot assay of Min6 cells treated with MG132, B Western blot assay of Min6 cells transfected with shFBW7, *p < 0.05 vs. the shControl group in FBW7 blotting, #p < 0.05 vs. the shControl group in EZH2 blotting. C RT-qPCR quantification of FBW7 and EZH2 mRNA in Min6 cells transfected with shFBW7, *p < 0.05 vs. the shControl group. D Western blot analysis of EZH2 after CHX treatment at different time points, EZH2 was first normalized to GAPDH then normalized to time 0 at each time point, *p < 0.05 vs. the shControl group. E After 48 h of transfection of designed plasmid, the Min6 cells were incubated with the MG132 (20 μM) for 8 h, Western blot analysis of the EZH2 ubiquitination following with IP assay. F Western blot assay of the FBW7 and EZH2 proteins in the FBW7-, or with EZH2-, overexpressed Min6 cells, *p < 0.05 vs. the normal group, #p < 0.05 vs. the OE-EZH2 + OE-NC group, &p < 0.05 vs. the OE-EZH2 + OE-FBW7 group. G CCK-8 assay for cell viability in each group, *p < 0.05 vs. the normal group, #p < 0.05 vs. the OE-EZH2 + OE-NC group, &p < 0.05 vs. the OE-EZH2 + OE-FBW7 group. H flow cytometry determination for apoptosis in each group, *p < 0.05 vs. the normal group, #p < 0.05 vs. the OE- EZH2 + OE-NC group, &p < 0.05 vs. the OE-EZH2 + OE-FBW7 group. I Determination of insulin secretion, *p < 0.05 vs. the normal group, #p < 0.05 vs. the OE-EZH2 + OE-NC group, &p < 0.05 vs. the OE-EZH2 + OE-FBW7 group. All experiments were conducted in triplicate. Data comparisons between two groups were analyzed by unpaired t-test. Comparisons among multiple groups were performed by one-way ANOVA. Statistical analysis in relation to time-based measurements within each group was realized using repeated-measures ANOVA.