Fig. 7

CYLD was regulated by CXCL12 and could deubiquitinate P62. (A) Venn plot for gene sets including CXCL12 H vs. L DEGs (upregulation), LC-MS/MS P62 result, deubiquitinases (DUBs) list and P62 interactors. (B) The expression of eight DUBs within 0–12 h after CXCL12 (50 ng/ml) stimulation detected by qPCR in T24 cells. (C) T24 cells were treated with CXCL12 for 0–12 h, detected by western blotting with CYLD, P62 and PDL1. (D) Western blotting showing the effect of CXCL12 (50ng/ml, 6 h) on CYLD, P62 and PDL1 in T24 cells with or without AMD3100 pretreatment (10ug/ml,12 h). (E) Western blotting showing the effect of knocking down CYLD on P62 and PDL1 with or without CXCL12 (50ng/ml, 6 h) stimulation in T24 cells. (F) Left: The degradation rate of P62 detected at the indicated time points by CHX chase assay (20µM) in T24 cells interfered by siCYLD. Right: Quantitative curve of the level of remained P62. (G) Co-immunoprecipitation (co-IP) showing the physical interaction between CYLD and P62 in T24 cells. (H, I) Western blotting showing the effect of knocking down CYLD on P62 ubiquitination in T24 and UMUC3 cells. (J) 293T cells transfected with the indicated plasmids for exogenous validation of the effect of CYLD on P62. (K) Western blotting showing the effect of CXCL12(50 ng/ml, 6 h) on CYLD, P62 and PDL1 with or without Stattic (5µM, 12 h) pretreatment in T24 cells. (L) T24 cells were treated with CXCL12 for 0–12 h, detected by western blotting with JAK2, pJAK2, CYLD, STAT3, pSTAT3, P62 and PDL1. (M) Schematic diagram showing the potential mechanism by which CAFs-secreted CXCL12 mediates PDL1 upregulation in bladder cancer