Figure 6. ARAF is required for the survival NRAS^Q61K-induced murine melanoma cell lines in absence of BRAF and CRAF.

(a) Growth curve analysis of resistant double knockout murine melanoma cell culture BRAF/CRAF^Δ/Δ (circles) compared to its parental control culture (triangles) for 6 days in presence of 10 μM U0126 (U0) or DMSO. Cell number is plotted relative to the initial number of cells at the start of treatment. Data are representative of three independent experiments. (b) Western blot analysis of ARAF, BRAF and CRAF protein expression and pERK activation in BRAF/CRAF^Δ/Δ and parental control cultures after treatment by 10 μM U0 or DMSO. Total ERK and β-actin are used as a loading control. (c) qRT–PCR analysis of ARAF expression in BRAF/CRAF^Δ/Δ and parental control cultures. ***P value<0.001 compared by Student's t-test. (d,f) Parental control and BRAF/CRAF^Δ/Δ cultures (d,f, respectively) were infected by lentiviruses encoding control shRNA or targeting ARAF (shARAF.1; shARAF.2; shARAF.3). After puromycin selection, cells were stained with crystal violet. (e,g) Western blot analysis of ARAF, BRAF and CRAF protein expression levels and pERK activation in parental control and BRAF/CRAF^Δ/Δ cultures (e,g, respectively) after infection by control or ARAF shRNA encoding viruses and selection. Total ERK and β-actin are used as a loading control. Control refers to Braf^f/f;Craf^f/f;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o parental culture and BRAF/CRAF^Δ/Δ refers to Braf^Δ/Δ;Craf^Δ/Δ;Tyr::NRAS^Q61K/^o;Ink4a^+/−;Tyr::CreERT2/^o double knockout culture. (h) Cell counting of parental control and BRAF/CRAF^Δ/Δ cultures infected with lentiviruses encoding control or ARAF shRNA on days 1, 6 and 10 after puromycin selection. (i) Proliferation rate in parental control and BRAF/CRAF^Δ/Δ cultures infected by lentiviruses encoding control or ARAF shRNA was measured after BrdU incorporation during 9 h. ***P value<0.001 compared by Student's t-test. ns, not significant. All data are represented as mean±s.d.