Research Paper Volume 14, Issue 18 pp 7527—7546

Figure 4. FAT10 regulates CHK1 expression through USP7. (A and B) Western blotting and qRT-PCR analyses of CHK1 expression levels in HK-2 cells stably transfected with shNC or shUSP7. (C and D) Western blotting and qRT-PCR analyses of CHK1 expression levels in HK-2 cells stably transfected with control vector or Flag-USP7. ^**P < 0.01. Tubulin was used as a loading control. (E) Knockdown or exogenous expression of USP7 altered the ubiquitination of CHK1 in HK-2 cells. The cells in each group were treated with MG132. (F) Western blotting of FAT10, CHK1 and USP7 in HK-2 cells stably transfected with Flag-FAT10 in the presence or absence of shUSP7. (G) Ubiquitinated CHK1 in HK-2 cells stably transfected with Flag-FAT10 in the presence or absence of shUSP7. The cells in each group were treated with MG132. (H and I) Upon hypoxia treatment, western blotting of FAT10, CHK1, CDKN1A, TGF-β and CTGF in FAT10-silencing HK-2 transfected with Flag-USP7 (H) or in FAT10-overexpression HK-2 transfected with shUSP7 (I). (J and K) Detection for cell cycle of FAT10-silencing HK-2 transfected with Flag-USP7 (J) or FAT10-overexpression HK-2 transfected with shUSP7 (K) following hypoxia injury. Results are expressed as peak diagram (left) and calculated distribution for cells in G2/M phases (right). ^*P < 0.05.