Fig. 3.

Identifying MYH9 as a HIF-1α-interacting protein and the effect of MYH9 on HIF-1α protein stabilization and ubiquitination. a Immunoprecipitated proteins of SNU-387 and SNU-387-LR cells were separated by SDS-PAGE and visualized by silver staining. b The top 10 protein scores from unique proteins that interact with HIF-1α were identified by LC-MS/MS analysis. c Western blot was conducted to assess the MYH9 expression in WT and HCC LR cells. d The interaction between endogenous MYH9 and HIF-1α was tested in Hep-3B-LR cells. IgG was used as a control. The MYH9 protein is indicated by the red arrow. e Colocalization of HIF-1α (green) with MYH9 (red) was observed by confocal microscopy in HuH-7 and HuH-7-LR cells. The nuclei were stained with DAPI. f Western blot analysis was used to assess the protein expression of MYH9 and HIF-1α following MYH9 knockdown (shMYH9) or shNC in HCC LR cells. g Western blot analysis was used to assess the protein expression of MYH9 and HIF-1α in WT HCC cells with MYH9 overexpression (MYH9) or vector. SNU-387-LR cells were treated with shMYH9 or shNC. h While Hep-3B cells were treated with MYH9 or vector (i). Both cell lines were then exposed to 10 μg/mL CHX for the designed time. The protein levels of HIF-1α and MYH9 were determined via western blot analysis. j SNU-387-LR and HuH-7-LR cells were treated with shMYH9 or shNC, and then exposed to MG132 (5 μM) for 2 hours. Western blot analysis was performed to assess the protein levels of HIF-1α and MYH9. k HuH-7-LR cells were cotransfected with shMYH9 or shNC and Ub (HA-tag) plasmids. l SNU-387 cells were cotransfected with MYH9 overexpression or vector and HA-Ub plasmids. Anti-HIF-1α antibody was used to isolate endogenous HIF-1α and anti-HA antibody was used to detect bound Ub. Exogenous MYH9 and HIF-1α expression in whole cell lysates was detected. m HuH-7-LR cells with shMYH9 or shNC were stably overexpressed with HIF-1α (wt) or mutated HIF-1α (P402A, P564A), HIF-1α (mut), which were with Flag-tag. Flag and MYH9 protein were determined by western blot, while the relative luciferase activity of HRE was analyzed (n). The data were presented as mean ± SD of three individual experiments. The Student’s t test was used for comparisons. **p < 0.01, ****p < 0.0001. Scale bar: 5 μm. IP immunoprecipitation, MYH9 non-muscle myosin heavy chain 9, WT wild type, LR lenvatinib resistant, HIF-1α hypoxia-inducible factor-1α, HCC hepatocellular carcinoma, CHX cycloheximide, HRE hypoxia-responsive element, ns nonsignificant