Fig. 7

ASAP2 binds to CIN85 to interrupt the CIN85-c-MET interaction and subsequent c-MET degradation. A Alteration of mRNA expression levels of c-MET from ASAP2 knockdown according to qRT-PCR. B shControl and ASAP2-knockdown SNU182 (left)/SNU387 (right) cells were serum starved for 12 h; Afterwards, the culture medium was replaced with serum-free DMEM with or without hepatocyte growth factor (HGF, 20 ng/mL) plus Cycloheximide (CHX, 50 μg/mL). Protein was extracted at the indicated time point, followed by western blot (WB) assays to evaluate the influence of ASAP2 knockdown on the half-life of c-MET protein. C shControl and ASAP2-knockdown SNU182 (left)/SNU387 (right) cells were serum starved for 12 h; Afterwards, the culture medium was replaced with serum-free DMEM with or without HGF (20 ng/mL) plus CHX (50 μg/mL); Protein was extracted at the indicated time point, followed by WB assays to evaluate the influence of ASAP2 knockdown on the p-AKT and p-ERK1/2 levels in HCC cells upon HGF stimulation. D Venn diagram demonstrating the overlapping ASAP2-interactors between the BioGRID and HIPPIE databases (upper), and HIPPIE scores for overlapping proteins (lower). E ASAP2 was pulled down, followed by WB assays to demonstrate the interaction between ASAP2 and CIN85 in HCC cells (upper); Then, CIN85 was pulled down, followed by WB assays to demonstrate the interaction between ASAP2 and CIN85 in HCC cells (lower). F The effects of ASAP2 on the interaction between CIN85 and c-MET in HCC cells were determined by immunoprecipitation (IP) experiments, followed by WB assays; HCC cells were treated with Bafilomycin A1 (BA, 100 nM) to prevent c-MET degradation as much as possible. G Increased abundance of exogenous ASAP2 expressed in Hep3B cells and its impacts on the interaction between endogenous c-MET and CIN85 were determined by IP followed by WB assays. H shControl SNU182 cells, ASAP2-knockdown SNU182 cells, ASAP2-knockdown SNU182 cells transfected with control siRNA, and ASAP2-knockdown SNU182 cells transfected with siCIN85 were serum starved for 12 h; Afterwards, the culture medium was replaced with DMEM containing 1% FBS with or without HGF (20 ng/mL) and the cells were cultured for 3 days; At day 3, Cell Counting Kit-8 (CCK8) assays were conducted to evaluate the proliferation rates. I shControl SNU182 cells, ASAP2-knockdown SNU182 cells, ASAP2-knockdown SNU182 cells transfected with control siRNA, and ASAP2-knockdown SNU182 cells transfected with siCIN85 were seeded into Transwell chamber inserts (8 μm pore size, upper chamber) at a density of 5 × 10⁴ cells per well; The upper chamber was supplemented with DMEM containing 1% FBS, while the lower chamber was supplemented with DMEM containing 1% FBS with or without HGF (20 ng/mL); Cells were cultured for 24 h to allow invasion; Afterwards, the upper chambers were collected and the invading cells were quantified with crystal violet staining to evaluate the invasion rates. J shControl SNU182 cells, ASAP2-knockdown SNU182 cells, ASAP2-knockdown SNU182 cells transfected with control siRNA, and ASAP2-knockdown SNU182 cells transfected with siCIN85 were serum starved for 12 h; Afterwards, the culture medium was replaced with serum-free DMEM with or without HGF (20 ng/mL) plus CHX (50 μg/mL), and the cells were cultured for 30 min; Total protein was extracted, followed by WB assays to determine the effects of ASAP2 knockdown on the levels of total c-MET, p–c-MET, p-AKT, and p-ERK1/2 of HCC cells upon HGF stimulation. “**” indicates 0.001 < P < 0.01