Fig. 4

Inhibition of autophagy degradation of PDL1 by CXCL12. (A) Differential expression pathways targeting GO and KEGG of GSVA analysis between high and low CXCL12 expression groups in TCGA. BP: biological process. CC: cellular component. MF: molecular function. (B, C) The effect of CXCL12 (50 ng/ml) on PDL1 expression with or without siCXCR4 or AMD3100 (10 µg/ml), detected by western blotting in T24 cells. (D) The expression of PDL1 within 0–24 h after CXCL12 stimulation detected by qPCR in T24 cells. (E) Left: The degradation rate of PDL1 detected at the indicated time points by CHX chase assay (20µM) in T24 cells treated with or without CXCL12 (50 ng/ml) stimulation. Right: Quantitative curve of the level of remained PDL1. (F) Western blotting of PDL1 ubiquitination stimulated by CXCL12 in T24 cells. (G) Detection of the effect of CXCL12 on autophagy and PDL1 in T24 cells by western blotting with PDL1, P62 and LC3. (H) After inducing autophagy in T24 cells using EBSS, the expression of PDL1 was detected by western blotting. (I) Immunofluorescence showing the T24 cells that transfected EGFP-mCherry-LC3 plasmid co-incubated with CXCL12 (50ng/ml, 6 h). Scale bars = 20 μm. (J) The effect of chloroquine (10 µM, 12 h) or rapamycin (1 µM, 12 h) on PDL1 expression and autophagy with or without CXCL12 (50ng/ml) stimulation detected by western blotting in T24 cells. (K) Immunofluorescence showing the colocalization between PDL1 and LC3 after CXCL12 stimulation (10 µM, 12 h) in T24 cells. (L) Western blotting showing the effect of knocking down ATG5 on PDL1 with or without CXCL12 (50 ng/ml, 6 h) stimulation in T24 cells