LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma
Fig 6
LMP1 co-expression with GLUT1 in NPC-LMP1 cells stabilized GLUT1 proteins by disrupting GLUT1 K48 ubiquitination and p62-dependent lysosomal degradation.
(A) LMP1 physically interacts with endogenous GLUT1. NPC-LMP1 and NPC-vector cells were treated with MG-132 (1 μM) for 4 h to stabilize GLUT1, followed by immunoprecipitation (IP) with an anti-GLUT1 antibody. Immunoblotting was performed using anti-LMP1 and anti-GLUT1 antibodies. Anti-IgG was used as a negative control for IP. Representative data from 5 independent experiments are shown. (B) Immunofluorescence detection of EBV-LMP1 and GLUT1 in stable NPC-LMP1 cell lines. NPC-LMP1 and NPC-vector cells were seeded onto a chambered cover glass overnight. LMP1 and GLUT1 were detected with LMP1 and GLUT1 antibodies, nuclei were stained with DAPI, and confocal images were analyzed using ImageJ software. (C) NPC-LMP1 and NPC-vector cell lines were treated with CHX for 18 h. Proteins were harvest at 0, 3, 6, 12 and 18 h, and the expression of GLUT1 was measured via immunoblotting. Representative data from 5 independent experiments are shown, and GAPDH was included as a control. (D) NPC-LMP1 and NPC-vector cell lines were treated with the lysosome inhibitor BafA1 for 12 h, proteins were harvested at 0 and 12 h, and GLUT1 expression was measured by immunoblotting. Representative data from 5 independent experiments are shown, and GAPDH was included as a control. (E) Immunoblotting showed that GLUT1 was expressed at higher levels in 293T cells than in p62KO-293T cells, whereas GLUT1 was expressed at higher levels in 293T-LMP1 cells than in 293T cells but was not found at higher levels in p62KO-293T-LMP1 cells compared with p62KO-293T cells. Representative data from 1 of 5 experiments are shown. (F-G). NPC-LMP1 and NPC-vector cell lines cultured in 6-well plates were transfected with hemagglutinin (HA)-tagged Ub (4 μg/well) (F) HA-tagged Ub-K48, HA-tagged Ub-K48R, HA-tagged Ub-K63 or HA-tagged Ub-K63R (G) and then treated with 20 mM MG132 for 6 h prior to harvest. Cell lysates were immunoprecipitated with anti-HA antibodies and then subjected to WB with an anti-GLUT1 antibody to measure the levels of ubiquitinated GLUT1 proteins (upper panels). WCLs were blotted to evaluate the levels of GLUT1 proteins (lower panels). β-actin expression was used as a protein loading control. The experiment shown is a representative of three independent experiments.