Fig 1.
Investigating the effects of the vemurafenib and erlotinib combination in BRAF and KRAS mutant NSCLC cells.
A: Western blot of phosphorylated AKT and ERK signaling in native untreated cell lines ([+] mutated; [–] wildtype). All lanes are from the same gel and break was created to include only relevant cell lines B: A549, H460, H1755 and HCC364 cell lines treated with D (DMSO) or indicated drugs, E (erlotinib), V (vemurafenib), and EV (erlotinib-vemurafenib) were analyzed after 72h by a MTS assay. The IC50 for Vemurafenib in HCC364 was 0.8 μM. C: Western blot of different NSCLC cell lines, showing changes in p-ERK, p-AKT and PARP with vehicle D (DMSO), E (erlotinib) 1.6 μM, V (vemurafenib) 1.6 μM, EV (erlotinib+vemurafenib) 1.6/1.6, μM after 24h treatment. D: Apoptosis by flow cytometry 48h post treatment with D (DMSO), E (erlotinib) 1.6 μM, V (vemurafenib) 1.6 μM, EV (erlotinib/vemurafenib 1.6/1.6 μM). Western blot, post 48h, supporting PARP cleavage with V and EV in HCC364 but not H1755 cells.
Fig 2.
Effects of single-agent vemurafenib on BRAF mutated NSCLC cells.
A: Long-term growth assay, 7 days post treatment with vehicle-D (DMSO), vemurafenib 50 nM, 500 nM, 5000 nM in both HCC364 and H1755 cells. HCC364 cells were more sensitive to vemurafenib. * p< 0.05; *** p<0.001 when compared to DMSO. B: Cell cycle analyses by flow cytometry 24h post treatment with DMSO, V (vemurafenib) in HCC364 and H1755 cells. Cell cycle phases shown are G1, S and G2 phase. V induces cell cycle arrest in HCC364 cells, as evidenced by an increase in G1 and a significant decrease in S phase. C: Western blot at 48 hours post treatment supporting the evidence for G1 arrest, showing increase in p27 and decrease in CDk2 with V (vemurafenib) when compared to D (DMSO). No effect was seen in H1755 cells. All lanes for HCC364 and all lanes for H1755 are from the same gel. The break has been created to remove erlotinib treated lanes.
Fig 3.
Growth and cell cycle effects of trametinib and vemurafenib combination on BRAF mutated NSCLC cell lines.
A, B: Long-term growth assay, 7 days post treatment with vehicle-DMSO (D), V (vemurafenib) 1 μM, T (trametinib) 1 μM and TV (trametinib + vemurafenib, 1 μM each) in HCC364 (A), and H1755 cells (B). C: Cell cycle analyses by flow cytometry in HCC364 and H1755 cells after 24 hours treatment with D, V 0.5 μM, T 0.5 μM, TV 0.5/0.5 μM. D: Western blot after 24h of H1755 and HCC364 treated like in C. (***p<0.001 when compared to DMSO).
Fig 4.
Anti-apoptotic effects of trametinib and vemurafenib in BRAF mutated NSCLC cell lines.
A: Apoptosis by flow cytometry, 48h post treatment with vehicle-D (DMSO), V (vemurafenib 1 μM), T (trametinib 1 μM), TV (trametinib + vemurafenib 1/1 μM) in HCC364 and H1755 cells. (*p<0.05;** p <0.01; *** p<0.001). B: Western blot of cleaved PARP in HCC364 and H1755, 48h post treatment with D (DMSO) and varying doses of vemurafenib (0.25, 0.5, 1μM) and trametinib (0.25, 0.5, 1μM) C: Western blot showing changes in ERK, AKT, BIM, PARP cleavage, 48h post treatment with vehicle D (DMSO), V (vemurafenib 1 μM), T (trametinib 1 μM), TV (trametinib + vemurafenib 1/1μM) in HCC364 and H1755 cells. Four lanes were treated in duplicates for each cell lines and only the left 4 lanes of each cell lines have been shown in the figure.