Systematic analysis of BRAFV600E melanomas reveals a role for JNK/c‐Jun pathway in adaptive resistance to drug‐induced apoptosis Covariate single‐cell.

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Systematic analysis of BRAFV600E melanomas reveals a role for JNK/c‐Jun pathway in adaptive resistance to drug‐induced apoptosis Covariate single‐cell analysis of Ki‐67 (left) and pRb(Ser807/811) (right) versus p‐cJun(Ser73) in WM1552C cells before and 24 h after exposure to 0.8 μM vemurafenib. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The vertical lines were used to gate p‐cJunHigh versus p‐cJunLow cells. The horizontal lines were used to gate Ki‐67High versus Ki‐67Low cells, and pRbHigh versus pRbLow cells. Analysis of drug dose‐dependent changes in proportion of pRbLow/p‐cJunHigh and pRbHigh/p‐cJunHigh subpopulations in four melanoma cell lines (WM115, WM1552C, LOXIMVI, COLO858) after exposure to vemurafenib for 24 h. These subpopulations were gated as shown in (A). Data are represented as mean ± SD for two replicates. Covariate single‐cell analysis of pRb(Ser807/811) versus pS6(Ser235/236) following 24‐h treatment of WM1552C cells with different doses of vemurafenib alone (top) and vemurafenib and JNK‐IN‐8 together (bottom). Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The horizontal and vertical lines were used to gate pS6High versus pS6Low cells, and pRbHigh versus pRbLow cells, respectively. Selected immunofluorescence images of pS6(Ser235/236) and Hoechst staining in WM1552C cells in a DMSO‐treated control and 24 h after exposure to 5 μM of vemurafenib, JNK‐IN‐8, and their combination. Analysis of the changes in proportion of pS6High cell population in three melanoma cell lines (LOXIMVI, WM115 and WM1552C) as a function of drug concentration for single‐drug vemurafenib and JNK‐IN‐8 treatments and their combination treatment. Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. pS6High population of cells was gated as indicated in (C). Data are represented as mean ± SD for two replicates. Data comparison between vemurafenib treatment and vemurafenib/JNK‐IN‐8 combined treatment was made by using two‐way analysis of variance (ANOVA). Fraction of c‐JunHigh cells (as measured by single‐cell immunofluorescence microscopy) after 48 h JUN knockdown followed by 24‐h treatment with vemurafenib. Fold changes are shown relative to control‐treated cells. Data are presented as mean ± SD. Single‐cell pS6(Ser235/236) levels in the c‐JunHigh and c‐JunLow fractions of cells (as measured by single‐cell multiplex immunofluorescence microscopy) after 48 h of JUN knockdown and 24‐h treatment with 0.32 μM vemurafenib. Single‐cell pS6 data are presented as box‐and‐whisker plots with median signal intensities and interquartile ranges; bars extending to 1.5× the interquartile range are shown for each condition as a measure of variance. P‐values were calculated using a two‐sided nonparametric Mann–Whitney U‐test. Source data are available online for this figure. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. Mol Syst Biol, Volume: 11, Issue: 3, First published: 26 March 2015, DOI: (10.15252/msb.20145877)