Fig. 4

LAMTOR1 inhibits exosomal PD-L1 by induced autophagy-lysosomal degradation. (A) Transmission electron microscopy (TEM) analysis revealed that LAMTOR1 facilitated an increase in the number of multivesicular bodies (MVBs) and their fusion with lysosomes in NSCLC cells (H1975 and H358). The scale bar in the insets is set at 500 nm. (B) Immunofluorescence studies demonstrated that LAMTOR1 induces the fusion of MVB (RAB7a) with autophagosome (ATG7) in NSCLC cells (H1975 and H358), with scale bars representing 10 μm. (C) Subsequently, NSCLC cells (H1975 and H358) co-transfected with OE-LAMTOR1 and si-RAB7a were assessed for the colocalization of PD-L1 with lysosomes (LAMP1), with scale bars representing 10 μm. (D) NSCLC cells (H1975 and H358) co-transfected with OE-LAMTOR1 and si-RAB7a were subjected to analysis of exosomal PD-L1 secretion through western blotting. NSCLC cells (H1975 and H358) were treated with OE-LAMTOR1 with or without si-RAB7a, and the right graph presents quantification of exosomal PD-L1 protein levels from six independent experiments. (E) Similar to previous setups, NSCLC cells (H1975 and H358) were treated with OE-LAMTOR1 with or without si-ATG7, and colocalization of PD-L1 with lysosomes (LAMP1) was evaluated. (F) Further experiments involved NSCLC cells (H1975 and H358) co-transfected with OE-LAMTOR1 and si-ATG7 for the analysis of exosomal PD-L1 secretion through western blotting. These results are represented as mean ± SEM from six assays, with significance levels indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on Student’s t test