Western blot (WB) analysis

For western blotting, total cell lysates were prepared as described previously [26]. The proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Amersham, Arlington Heights, USA). Membranes were probed with primary antibodies and the signal was detected using peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson Immunoresearch Labs, Inc., Baltimore, USA). The enhanced chemi-luminescence (ECL) system (Amersham) was used for detection. The following primary Antibodies (Abs) were used: phosphorylated (Thr202/Tyr204) and total ERK1/2, BRAF, phosphorylated (Tyr1173), phosphorylated (Tyr1068) and total EGFR, phosphorylated (Tyr1248) and total HER2, phosphorylated (Ser217/221) and total MEK1/2, phosphorylated pP90^RSK (Ser380) (from Cell Signaling Technology Inc. Beverly, USA) and CRAF (from Santa Cruz Biotechnology, Santa Cruz, CA). To control the amount of proteins transferred to nitrocellulose membrane, β-actin, Hsp70 and Tubulin were used and detected by anti β-actin mAb (clone AC-15, Sigma, St. Louis, USA), anti Hsp70 mAb (from Calbiochem Merck Biotechnology), and anti-Tubulin mAb (from Abcam, Cambridge, MA, USA). Image detection was performed with Amersham Hyperfilm ECL (Amersham, Amersham, Chicago, IL; Figs. 1, ​,4,4, ​,7;7; Additional file 1: Fig. S1). For the organoids, protein lysates were fractionated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane and then blocked with 5% BSA in TBST (1% Tween 20, tris-buffered saline). Proteins shown in Fig. 5 were detected on Kodak films (Sigma-Aldrich, St. Louis, MO), using HRP-conjugated secondary antibodies.

Molecular analysis in KRAS-mut lung and pancreatic cell lines. a-d. Lung cancer cell line A549 and pancreatic cancer cell line MiaPaCa2 were treated with dabrafenib (a) and RAF265 (b) alone or in combination with trametinib (c-d) for 4 h at the indicated doses. The cells were lysed and analyzed by Western Blotting using antibodies specific for the indicated proteins. Western blot with Hsp-70 specific antibody is shown as protein loading and blotting control. e A549 cells were treated with dabrafenib (10 μM) or RAF265 (10 μM) for 4 h. Endogenous CRAF was immunoprecipitated and the immunocomplexes were blotted for BRAF or CRAF. BRAF and CRAF levels in total cell lysates are also shown

Effects of single and combined MEK and RAF inhibition in LCSC. a and c. Cells obtained from lung cancer spheres (LCSC) dissociation were treated with trametinib (10 nM) and dabrafenib (10 μM) alone or in combination for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the protein above indicated. Western blot with Tubulin specific antibody is shown as protein loading and blotting control. b and d. LCSC2 and LCSC3 cells were plated in 96-well flat-bottom plates; trametinib and dabrafenib were added at their final concentration (1–10 nM), as single agents or in a fixed dose-ratio combination (1:1000). The results represent the average ± SD of three independent experiments. CI were calculated by conservative isobologram analysis for experimental data and plotted against the fraction affected

Effects of single and combined MEK and RAF inhibition in patient-derived pancreatic organoids. a. Phase-contrast images showing an organoid formation assay. Organoids were seeded as singles cells and treated after 48 h with different doses of trametinib (10 nM,), dabrafenib (10 μM) and combination ratio 1:1000. Images were detected with EVOS Cell Imaging System (b). Patient-derived pancreatic organoid T1 (KRAS-mut) was incubated with dabrafenib and trametinib alone or in combination for 24 h. Protein lysate were analyzed by Western Blotting using p-ERK antibody. β-Actin is used as protein loading at blotting control. c. Patient-derived pancreatic organoid T1 was treated with increasing amounts of the dabrafenib (0.1–10 μM) alone or in combination with trametinib (01–10 nM) ratio 1:1000, for 72 h. Cell viability was evaluated by Cell Titer Glo 2.0 assay. The table shows the CI of trametinib and dabrafenib in a normal pancreatic organoid N1 and in 4 different PDAC organoids KRAS-mut

Selective BRAF inhibition induces EGFR family-dependent MAPK hyperactivation in Calu-3 cells. a and b The NSCLC cell line Calu-3 (HER2-amplified, KRAS-wt) was treated with increasing concentrations of dabrafenib (0.01–10 μM) alone a or in combination with trametinib (ratio 1:1000; b) for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the proteins indicated. Western blot with Hsp70 specific antibody is shown as protein loading and blotting control. c Calu-3 cells were treated with increasing concentrations of dabrafenib (0.01–10 μM) and trametinib (0.01 nM–10 nM) alone or in combination for 72 h. Cell viability was assessed by Crystal violet assay and pharmacologic interactions were evaluated using the Calcusyn software. The results represent the average ± SD of three independent experiments. d Calu-3 and HCC827 (EGFR-mut) cells were treated with increasing concentrations of dabrafenib (0.1–10 μM) and lapatinib (0.1–10 μM) for 4 h. The proteins were subjected to Western Blotting and analyzed for the indicated antibodies. e MiaPaCa2, A549 and A427 cells were treated with dabrafenib (10 μM) and lapatinib (10 μM) alone or in combination for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the protein above indicated