Fig. 1: SRC is activated following BRAF inhibition in BRAFm CRC cell lines and efficiently inhibited by dasatinib.

a On the left, proteome human phospho-kinase array of HT29 cell line following vemurafenib treatment (IC50 value, 24 h). The black boxes in the figure highlight pSRC (Y419) upregulation, and the pixel densitometry representation of this data is shown below. On the right, expression of SRC and pSRC in the panel of CRC BRAF^V600E cell lines treated with BRAFi (vemurafenib (HT29, SW1417 and WiDr) or encorafenib (LS411N and RKO)) at their IC50 values for 8 h. SRC activation is reflected by increased phosphorylation of the SRC activation site Y419 (pY419). β-actin used as a loading control. Molecular weight/size markers are indicated on the right (kDa). Images of WB are representative of 3 independent experiments. b Dose-response curves of HT29 cells (SRC OE and SRC KO cells) after treatment with vemurafenib or encorafenib for 72 h (left). On the right, representation of IC50 values of modified HT29 cells after treatment with vemurafenib or encorafenib for 72 h, compared to controls. Data represent the mean ± SEM of at least 3 biologically independent experiments. c Representative WB analysis of pSRC and SRC in HT29 cells after being treated at different concentrations (0.2–2 µM) for 24 h and with 1.2 µM dasatinib at the indicated time points (1–24 h), and their respective controls. β-actin used as a loading control. Data are representative of 3 independent experiments. Protein quantification is shown below as a pSRC/SRC ratio. d Dose-response assay of HT29 treated with dasatinib for 72 h, after treatment with the IC20 (0.06 µM), IC50 (1.20 µM) and IC70 (4.11 µM) doses (n = 3 replicates examined). e Transwell assay for cell migration in HT29 cells after being treated with dasatinib or control (DMSO) for 24 h. Transmigrated cells were quantified by cell titter. Error bars represent the mean ± SD of 4 biologically independent experiments. Significance was considered for *P < 0.05, **P < 0.01 and ***P < 0.001. D dasatinib, IC20 20% inhibitory concentration, IC50, half-maximal inhibitory concentration, IC70 70% inhibitory concentration, CTRL control, OE overexpression, KO knock-out.