Fig. 3: RNF8 overexpression impairs GSC mitotic progression that is dependent on its FHA and RING domains.
From: CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to induce mitotic checkpoint in glioma
A Western blot analysis of RNF8 and SAC markers, including H3 pS10 and cyclin B1, in GSCs transduced with different RNF8 constructs. RNF8 serves as the positive control whereas GAPDH and β-actin serve as loading control respectively. B Tumorsphere formation of two GSC lines overexpressing GFP, RNF8, *FHA, or *RING (n = 6) (mean±SD). *P < 0.05; **P < 0.01. C, D Colony formation of two GSC lines overexpressing GFP, RNF8, *FHA, or *RING (n = 4) (mean ± SD). *P < 0.05; ***P < 0.001. D Representative images of (C). E Cell cycle analysis of TS543 overexpressing GFP, RNF8, *FHA or *RING, with 40 µM Z-VAD-FMK treatment (72 h) (n = 3) (mean±SD). ***P < 0.001. F Western blot analysis of MAD2 levels in the RNF8 IP from RNF8 overexpressing GSC TS543 lysates. G Western blot analysis of H3 pS10 and cyclin B1 levels of GSC TS543 with GFP or RNF8 overexpression, with or without MAD2 depletion. RNF8 and MAD2 serve as the positive controls, while GAPDH serves as the loading control. H, I Karyotyping analysis of GSC TS543 overexpressing different RNF8 constructs (n = 3). Near triploidy: 60–80 chromosomes; near tetraploidy: 81–110 chromosomes. Minimum of 100 spreads were analyzed per condition. I Representative karyotypes from GSCs overexpressing different RNF8 constructs (H). Scale bar 20 μm.
