Fig. 7.

p-MYH9 (Ser1943) recruits USP22 to deubiquitinate HIF-1α and promote LR. a HuH-7 cells with vector and MYH9 overexpression and HuH-7-LR cells, accompanied by USP22 knockdown (shUSP22) or not, the altered protein expression of MYH9, HIF-1α and USP22 was detected through western blot. b Both the shUSP22 and Ub (HA-tagged) plasmids were transfected into HuH-7 cells with MYH9 overexpression and HuH-7-LR cells. Anti-HIF-1α antibody was used to isolated the HIF-1α protein and anti-HA antibody was used to detected bound Ub. MYH9, HIF-1α and USP22 protein expression levels were determined in whole cell lysates. c The interaction of endogenous MYH9, HIF-1α and USP22 was detected in HuH-7-LR cells. IgG was used as a control. d USP22 (Flag-tag) plasmid was transfected with MYH9-S1943E (myc-tag) or MYH9-S1943A (myc-tag) plasmids in HEK-293T cells. The interaction of myc protein and Flag protein was detected. IgG was used as a control. e HuH-7-LR cells were transfected with shNC or shUSP22, and cells were treated with DMSO or 2 μg/mL USP22 inhibitor, S02 combined with the indicated concentration of lenvatinib. Variations in lenvatinib and USP22 inhibitor sensitivity were detected by trypan blue staining-based cell count. The data were presented as mean ± SD of three individual experiments. f HuH-7-LR cell xenografted nude mice were fed lenvatinib (30 mg/kg), S02 (10 mg/kg) and their combination daily for three weeks after the tumor volume reached an average size of 50–100 mm³ (n = 6 per group). The isolated tumors were photographed. g Tumor volume of each group was measured twice a week. After 21 days, the mice were sacrificed. The data were presented as mean ± SEM. h Immunohistochemistry was used to detect the expression of HIF-1α and Ki67 in the xenografts after various treatments. i Graphical summary of the molecular mechanisms involving MYH9, HIF-1α and USP22 in regulating of CSC properties in lenvatinib-resistant cells. The graph was created by Microsoft Powerpoint. The Student’s t-test was used for comparisons. *p < 0.05, ***p < 0.001. Scale bar: 100 μm. MYH9 non-muscle myosin heavy chain 9, LR lenvatinib resistant, HIF-1α hypoxia-inducible factor-1α, USP22 ubiquitin-specific protease 22, DMSO dimethyl sulfoxide, CSC cancer stem cell