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. 2014 Feb 3;111(7):E748–E757. doi: 10.1073/pnas.1320956111

Fig. 2.

Fig. 2.

A switch to p61VE is necessary and sufficient to drive BRAF-inhibitor resistance in the NSCLC models. (A) Expression of full-length (FL) or aberrant (p61) BRAF shown as fragments per kilobase of exon per million fragments mapped (FPKM) in each HCC364 cell line. (B) RNA sequencing reads that reflect splicing among exons is shown in VR1–VR5 lines demonstrating aberrant splicing of exons 3 and 9 indicated the absence of exons 4–8 in the VR1 and VR2 sublines only. Line widths reflect relative expression levels and in VR1-VR2 mirror the results in A. (C) Western blot analysis of each indicated protein in lysates from the HCC364 parental cell line and each isogenic vemurafenib-resistant subline (VR1–VR5). Data represent three independent experiments. (D) Western blot analysis of each indicated protein in lysates from HCC364 parental cells and from the isogenic vemurafenib-resistant subline VR1 transfected with p61 and full-length BRAF-specific siRNA. Data represent three independent experiments. (E) Effect of each indicated siRNA on the viability of HCC364 VR1 cells. Data shown (±SEM) are normalized to the vehicle treatment (n = 3). (F) Dose–response curve showing the effect of vemurafenib on the viability of HCC364 parental cells overexpressing either empty vector or p61VE plus either nontargeting siRNA (control) or BRAF siRNA targeting only full-length BRAF. Data are shown as ± SEM (n = 3). (G) Western blot analysis (Upper) and quantification (Lower) of each indicated protein in lysates from HCC364 parental cells overexpressing empty vector or p61VE plus either siRNA or BRAF siRNA targeting only full-length BRAF. Data represent three independent experiments. (H) (Upper) Relative cell number of HCC364 VR1 cells after transient transfection of empty vector, wild-type BRAF, or BRAF-V600E (BRAF-VE) constructs and treatment with 2 µM of vemurafenib. (Lower) Western blots for the indicated proteins in lysates from the VR1 cells analyzed. All cell-viability data shown are normalized to the control siRNA or vehicle treatment (n = 3, *P < 0.05).