Fig. 4

CircMET evaluation reveals MET-driven acquired resistance and mirrors MET-targeted therapy response in vitro. a Gene Copy Number (GCN) analysis of the indicated parental (par.) and clonal (cl.), sensitive and resistant (res.) HCC827 cell populations. Red dashed line indicates the twofold threshold for MET amplification. b Dot plot of circMET levels measured by real-time PCR in 11 among HCC827 and WiDr parental and resistant cells with and without MET amplification. Triangles are referred to WiDr parental and resistant cells. c Proliferation assay of afatinib-resistant HCC827 clonal cells treated with the indicated inhibitors. Representative pictures are shown below (scale bar = 500 μm). d Soft-agar colony formation assay of afatinib-resistant HCC827 clonal cells treated with the indicated inhibitors. Representative pictures are shown below (scale bar = 500 μm). e, f MET GCN (e) and real-time PCR analyses (f) of afatinib-resistant HCC827 cell subclones upon 7 days of treatment with the indicated inhibitors. AFAT, afatinib; ERLOT, erlotinib; CTX, cetuximab. Data are expressed as mean ± SEM. ***P < 0.001, Student’s t test