Fig. 2

CircMET RNA characterization. a PCR analysis of the indicated transcripts. MET Δex2 represents the 7-Kb MET transcript lacking exon 2. b Schematics of primer pairs designed to amplify linear MET exon 2 (convergent) or its deriving circMET RNA product (divergent). c PCR analysis on genomic DNA (gDNA) and complementary DNA (cDNA) derived from the indicated cell lines. Junction-Spanning (JS) divergent primers were used to specifically detect circMET, while convergent primers were used to detect linear MET mRNAs. ACTB was used as a control. d Schematic representation of the probe used for Northern blot analysis together with its location on the linear and circular MET RNAs and Northern blot on 7.5 µg total RNA from GTL16 cell line treated or not with RNase R. The linear and the circular RNA forms are indicated next to the gels with the ‘‘–’’ and ‘‘o’’ symbols, respectively. e Real-time PCR analysis of linear MET and circMET levels upon RNase R digestion in the indicated cell lines. f Real-time PCR analysis of circMET turnover in the indicated cells treated with Actinomycin-D (n = 5). g-i Real-time PCR analysis of circMET and linear MET mRNA levels during myogenic differentiation of RD18 human rhabdomyosarcoma cells conditionally expressing miR-206 (g), NIH 10T1/2 murine fibroblasts conditionally expressing MyoD (h), and murine muscle stem cells in proliferation and differentiation medium (i). j Quantification of circMET levels in nuclear and cytoplasmic fractions of the indicated cell lines. RNU48 was used as a nuclear positive control. Data are expressed as mean ± SEM. ^NSP > 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test