Fig 1: CD73 generates adenosine in MET-amplified EGFR-TKI–resistant cells and is regulated by FRA1. A and B, Immunoblot of the phospho- and total FRA1 proteins or qRT-PCR of FRA1 in HCC827 and HCC827GR6 cell lines or HCC827GR6 cell lines with Scramble (Scr) or MET sgRNA. Mean ± SEM of n = 3 biological replicates is shown (unpaired two-tailed Student t test). C, Immunoblot of indicated proteins in HCC827 cells overexpressing LUC or FRA1. D and E, CD73 expression on HCC827GR6 or DFCI-202 cells with Scramble or FRA1 sgRNA. F, Immunoblot of indicated proteins HCC827GR6 cell lines treated with indicated compounds for 48 hours. OSI, osimertinib; SAVO, savolitinib; TRAM, trametinib. G, Adenosine assay protocol using Adenosine Assay Kit (Abcam, catalog no. ab211094). H, Immunoblot of indicated proteins in HCC827GR6 cells with Scramble or CD73 sgRNA. Adenosine levels in CM derived from HCC827GR6 cells with Scramble or CD73 sgRNA for 72 hours. Mean ± SEM of n = 3 biological replicates is shown (unpaired two-tailed Student t test). I, Immunoblot of indicated proteins in HCC827 cells overexpressing LUC or CD73. Adenosine levels in CM derived from HCC827 cells overexpressing LUC or CD73 for 72 hours. Mean ± SEM of n = 3 biological replicates is shown (unpaired two-tailed Student t test). J, Adenosine levels in CM derived from HCC827 or HCC827GR6 cells for 72 hours. Mean ± SEM of n = 3 biological replicates is shown (unpaired two-tailed Student t test). K, Adenosine levels in CM derived from HCC827GR6 cells with Scramble or MET sgRNA for 72 hours. Mean ± SEM of n = 3 biological replicates is shown (unpaired two-tailed Student t test). *, P < 0.05; **, P < 0.005; ***, P < 0.001.