Figure 6
From: JUN dependency in distinct early and late BRAF inhibition adaptation states of melanoma
Co-targeting mutant BRAF signaling and JUN function results in synergistic death and diminishes the persistent cell reservoir. (a–d) Effect on cell viability of 1 μm vemurafenib and JUN short interfering RNA (siRNA) knockdown co-treatment for parental (P), fully resistant (R), up-front co-treated (+vem(3d)), and short-term persisting cell (+vem(6d)). Viable cells were counted by Trypan-blue staining with a ViCell counter. A JUN siRNA pool was used for the experiments in this figure, see Supplementary Table S12 for confirmation with individual siRNAs and Supplementary Table S13 for confirmation with distinct JUN short hairpin RNAs (shRNAs). (e–h) Induction of apoptosis (Caspase-3/7 activation) in melanoma cells co-treated with 1μm vemurafenib and JUN siRNA. (i–l) Western blot of melanoma cells co-treated with 1μm vemurafenib and JUN siRNA. PARP cleavage is shown as an apoptosis marker. Phospho-JUN was included for M249P cells owing to its lower upregulation of total JUN (see Figure 5g). Significant pairwise differences with a t-test P-value <0.05 are indicated (*). Overall, JUN siRNA knockdown had a significant effect on cell viability both for the panel of resistant (P-value=4E−6) and persisting cells (P-value<2E−16) and significantly induced apoptosis in the resistant/persisting cell line panel (P-value=1E−8), but not in the parental cell line panel (two-way analysis of variance P-values with JUN siRNA treatment and cell line as factors; significance level α=0.01).
