Fig. 5
P62 mediated autophagic regulation of PDL1 by CXCL12. (A) Correlation between CXCL12, CXCR4, P62 and PDL1 in TCGA bladder cancer data. The coordinate axis represents log2 (gene expression). (B) Western blotting showing the effect of knocking down CXCR4 on P62 and PDL1 with or without CXCL12 (50 ng/ml, 6 h) stimulation in T24 cells. (C) The effect of CXCL12 (50 ng/ml, 6 h) stimulation on P62 and PDL1 expression was examined by western blotting after pretreatment of T24 cells using different concentrations of AMD3100. (D) Western blotting showing the effect of CAF-CM (12 h) on P62 and PDL in T24 cells with or without AMD3100 pretreatment (10ug/ml,12 h). (E) After knockdown of CXCL12 in CAFs, its culture medium (CM, 12 h) was used to stimulate T24 cells and then CXCL12 (50 ng/ml, 6 h) was added to rescue. Western blotting was used to detect changes of P62 and PDL1. (F) Western blotting showing the effect of knocking down P62 on PDL1 with or without CXCL12 (50 ng/ml, 6 h) stimulation in T24 cells. (G) Left: Compare the effect of CXCL12 (50 ng/ml, 0-12 h) on PDL1 and LC3 between the P62 knockdown and control groups of T24 cells by western blotting. Right: Quantitative curves of protein changes of PDL1 and LC3. (H) Co-immunoprecipitation (co-IP) showing the physical interaction between PDL1 and P62 in T24 cells. (I) Co-IP showing the effect of CXCL12 (50 ng/ml, 6 h) on the interaction between PDL1 and P62 in T24 cells. (J) Left: Immunofluorescence showing the colocalization between PDL1 and P62 after stimulation with CXCL12 (50 ng/ml, 6 h) in T24 cells. Scale bar = 10 μm. Right: The statistical results of colocalization (Pearson’s R value). Values are means ± SD from three independent experiments. ** P < 0.01; *** P < 0.001; **** P < 0.0001