Fig. 3: Hsc70 competes with CMTM6 for binding and regulation of PD-L1.

A, B MCF-7 cells were transfected with siRNA of Hsc70 and CMTM6 for 60 h, PD-L1 levels were detected by western blotting (A), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry (B). C HEK293T cells were transfected with siRNA of CMTM6 for 48 h, then transfected with PD-L1-Flag for another 12 h, the interaction between Hsc70 and PD-L1 was detected by immunoprecipitation. D, E MCF-7 cells were transfected with siRNAs of CMTM6 and TSG101 for 60 h, cell lysates were immunoblotted with indicated antibodies (D), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry, n = 6 (E). F, G MCF-7 cells were transfected with siRNAs of CMTM6 and VPS4 for 60 h, cell lysates were immunoblotted with indicated antibodies (F), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry, n = 6 (G). H, I MCF-7 cells were transfected with siRNA of CMTM6 for 48 h, treated with or without U18666A (5 μg/mL) for another 12 h, PD-L1 levels were detected by western blotting (H), fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry, n = 6 (I). J–L MCF-7 cells were transfected with indicated siRNAs for 36 h and the co-localization between RAB7A and PD-L1 (J), the co-localization between LAMP1 and PD-L1 (K), the co-localization between RAB11 and PD-L1 (L) were done using immunofluorescence and confocal microscopy. Scale bar, 5 μm. The intensity profiles along the yellow line are plotted in the middle panels, with the colocalizing sites marked by white. For (B, E, G, I), MCF-7 cells were seeded in 48-well plate with 6 replicates per group and subjected to the corresponding treatment, repeated independently three times and similar results were obtained. Data shown in (A), (C), (D), (F), (H), and (J–L) were repeated independently three times with similar results. Data represent Mean ± SEM, for (E, G, I) data, two-sided with adjustment of Tukey’s multiple comparisons, one-way ANOVA, P value is indicated in the graph. The numbers under blots represent the value (the ratio to Tubulin/Flag) of grayscale quantification. Source data are provided as a Source data file.