Fig. 1: EGFR inhibitors were screened to reduce membrane PD-L1 levels.
From: ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation
a High-throughput screening of 2125 FDA-approved drugs or drug candidates. U937 cells were incubated with IFNγ (100 ng/mL) for 48 h, treated with the drugs at 10 μM for 12 h. Ruxolitinib (Rux) was used as a positive control. The hit compounds that induced the decrease of PD-L1 levels are shown in blue. The depth of blue represents decreased level of PD-L1. The heatmap represents the targeted pathways obtained from the high-throughput screening, based upon decreased membrane PD-L1 level detected by flow cytometry. b Immunoblotting of PD-L1 in U937 cells treated with ES-072 (ES) or AZD9291 (AZD) at indicated concentrations for 12 h. ES and AZD are EGFR inhibitors. c, d Median fluorescence intensity (MFI) (c) and relative quantification (d) of PD-L1 in U937 cells treated with 10 μM ES-072 or 10 μM AZD9291 for 12 h. Data represent means ± SEM, n = 18, 6 independent repeats, ****P < 0.0001. e–g Immunoblotting (e, f) and flow cytometry (g) analysis of PD-L1 levels in U937 cells treated with 10 μM ES-072 or 10 μM AZD9291 for the indicated times. h, i Immunoblottings (h) of PD-L1 and β-TrCP, relative quantification (i) of PD-L1 MFI in H1975 cells transfected with non-targeting siRNA (si-CTRL) or β-TrCP siRNAs (si-β-TrCP) and treated with or without 10 μM ES-072/AZD9291 for 12 h. Data represent means ± SEM, n = 9, 3 independent repeats, NS: no significant; ****P < 0.0001. j–l Immunoblotting (j) and flow cytometry analysis (k) with relative quantification (l) of PD-L1 in H1975 cells treated with 10 μM ES-072 or AZD9291, and/or 5 μM LY2090314 (LY) for 12 h. LY is a GSK3 inhibitor. Data represent means ± SEM, n = 9, 3 independent repeats, ****P < 0.0001. Source data are provided as a Source Data file.
