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. 2022 Aug 3;83:104200. doi: 10.1016/j.ebiom.2022.104200

Figure 5.

Figure 5

EGFR 19Del/T790M-cis-L792F stimulated JAK/STAT3/IL-4 signaling. (a). Heatmap of cytokine antibody array in H1975 control and H1975 L792Fcis cells. (b-c). IL-4 expression was detected by western blot (b) and ELISA analyses (c) in the H1975 control, H1975 L792Fcis, PC-9 control, and PC-9 L792Fcis cells (n=3, p=0.016 or 0.007, respectively. Statistical differences were evaluated using t test). (d). Signal pathway enrichment in the H1975 L792Fcis cells compared to the H1975 control cells. (e). Western blot shows ERK, p-ERK Thr202/Tyr204, AKT, p-AKT Ser473, JAK, p-JAK Tyr1034/1035, STAT3, p-STAT3 Tyr705, p-STAT3 Ser727, STAT6, and p-STAT6 Try641 expression in the H1975 control, H1975 L792Fcis, PC-9 control, and PC-9 L792Fcis cells. (f). Schematic depiction of the IL4 promoters (−1000 to 1 base pair, black line) with STAT3 transcription factor binding sites (TFBs). (g-h). ChIP assay using p-STAT3 antibody and IgG as internal controls. PCR (g) and RT-qPCR (h, n=3, p=0.006, statistical differences were evaluated using t test.) were performed to amplify p-STAT3 TFBs of IL4 promoter in the ChIP product. (i). IL4 promoter activity of luciferase reporter in the PC-9 control and PC-9 L792Fcis cells with wild-type or mutant p-STAT3-binding sequences (n=3, p=0.00001 or 0.00002, statistical differences were evaluated using t test). PRL-TK plasmid was used as an internal control. Data are presented as means ± standard deviation.