Fig. 6: AUY-922 promotes PD-L1 degradation through Hsc70-mediated eMI.
From: Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation
A Structural formula of AUY-922. B MCF-7 cells were treated with 1 μM AUY-922 for 0, 2, 4, 8, and 12 h. PD-L1 levels were detected by western blotting. C MCF-7 cells were treated with 1 μM AUY-922 for 6 h and 12 h, fluorescence of PD-L1 on the cell membrane was analyzed by flow cytometry, n = 6. D MCF-7 cells were treated with 1 μM AUY-922 for 12 h, treated with or without MG132 (10 μM), NH4Cl (20 mM), Leupeptin (100 nM), 3-MA (5 mM), E-64D (10 μM) for another 12 h. Cell lysates were detected by western blotting. E, F MCF-7 cells were transfected with siRNAs of Lamp2a for 48 h, treated with or without 1 μM AUY-922 for another 12 h. Fluorescence of PD-L1 on the cell membrane was analyzed by flow cytometry, n = 6 (E), cell lysates were detected by western blotting (F). G MCF-7 cells were transfected with siRNAs of TSG101 for 48 h, treated with or without 1 μM AUY-922 for another 12 h. Fluorescence of PD-L1 on the cell membrane was analyzed by flow cytometry, n = 6. H MCF-7 cells were transfected with siRNAs of VPS4 for 48 h, treated with or without 1 μM AUY-922 for another 12 h. Fluorescence of PD-L1 on the cell membrane was analyzed by flow cytometry, n = 6. I MCF-7 cells were treated with 1 μM AUY-922 with or without 5 μg/mL U18666A for 12 h. Fluorescence of PD-L1 on the surface of cell membrane was analyzed by flow cytometry, n = 6. J–L MCF-7 cells were treated with 1 μM AUY-922 for 4 h and the co-localization between RAB7A and PD-L1 (J), LAMP1 and PD-L1 (K), 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 (C, 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 (B, D, F) and (J–L) were repeated independently three times with similar results. Data represent Mean ± SEM, for (C, E, G–I), n = 6, 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) of grayscale quantification. Source data are provided as a Source data file.
