Figure 2.

MET blockade drives PDL1 expression by suppression of GSK3B-mediated PDL1 degradation in HCC cells. (A) Western blot analysis of Flag-PDL1 expression using Flag antibody in MET-knockdown Hep3B cells transfected with Flag-PDL1. (B) Immunoblot analysis of whole cell lysates derived from vector control or MET-overexpressing Hep3B cells given MG132 (5 μmol/L) for 16 hours and LiCl (25 μmol/L) for 5 hours. (C) Ubiquitination assay of PDL1 in MET-knockdown Hep3B cells. Ubiquitinated PDL1 was immuno-precipitated and subjected to western blot analysis with antibody against ubiquitin. Cells were treated with MG132 before ubiquitination analysis. (D) Stability of PDL1 protein in Hep3B cells transfected with HA-PDL1 and WT Flag-MET or MET-KD. Cells were treated with CHX 20 mmol/L at the indicated intervals and subjected to western blot analysis. (E) Quantification of PDL1 half-life in indicated groups. (F) Kinase activity of GSK3B in MET-knockdown Hep3B and SK-HEP-1 cells according to an in vitro kinase assay and phosphorylation analysis. Columns indicate mean activity after subtraction of background phosphorylation. **P < .01. (G) Western blot analysis of PDL1 phosphorylation at T180 and S184 by phospho-Y180 and phospho-S184 PDL1 antibodies in vector control and MET-knockdown Hep3B cells, respectively. (H) Coomassie blue staining of GSK3B-interacting proteins in Hep3B cells. Interacting proteins were isolated and identified by mass spectrometry. (I) Endogenous co-immunoprecipitation of Hep3B cells using MET and GSK3B antibodies. Cell lysates were analyzed by western blotting. (J) In vitro assay of GSK3B phosphorylation by MET at Y56. Purified WT GST-GSK3B and Y56F were incubated with recombinant MET kinase in the presence of adenosine triphosphate at 30°C for 30 minutes. Protein lysates were analyzed by western blotting. (K) (Left) Kinase activity of GSK3B in Hep3B and SK-HEP-1 cells expressing pMX (empty vector), WT GSK3B, or GSK3B Y56F by in vitro kinase assay and phosphorylation analysis. **P < .01. (Right) immunoblot of PDL1 and GSK3B expression in Hep3B cells transiently transfected with pMX (empty vector), WT GSK3B, and/or GSK3B Y56F. (L) (Top) Schematic of drug intervention protocol for PD1 antibody in C3H mice. At the drug intervention end point, tumors were isolated for immunofluorescent analysis. (Bottom) Growth of HCA-1 tumors in C3H mice that were treated with or without the PD1 antibody. Tumors were measured at the indicated time points. CHX, cycloheximide; CTRL, control; E.V., empty vector; GST, glutathione S-transferase; HA-PDL1, hemagglutinin-tagged PDL1; IgG, immunoglobulin G; IP, immuno-precipitated; KD, kinase-dead; OE, overexpression.