Fig 1: Myogenic induction of DUX4 and DUX4 target genes in FSHD cells with genetic depletion or pharmacological inhibition of p38α/β (MAPK14/11). (A) Deletion of the p38 genes by CRISPR/Cas9 editing in FSHD cells. Western blot analysis of lysates generated from p38 MAPK CRISPR knockouts. Bands for MAPK14 and MAPK11 are shown in parental (Unmodified), Cas9 expressing (Cas9) or genome-edited cell lines with deletion of p38α (MAPK14-/-), p38β (MAPK11-/-) or p38α/β (MAPK14/11-/-). α-Tubulin expression is detected to ensure equal loading. (B) Relative RNA levels for DUX4 and DUX4 targets ZSCAN4, MBD3L2, and LEUTX in differentiating FSHD1 (FSHD1_Cas9), FSHD2 (FSHD2_Cas9) and corresponding double KO lines (_MAPK14/11-/-) at the indicated time points (72 and 120 h) after initiating myogenic differentiation. (C) Relative DUX4 and DUX4 target RNA levels in FSHD1 and FSHD2 myocytes treated with vehicle (_Unmod) or 1 μm losmapimod (_LOS) during a 120-hour differentiation period. Myoblasts in growth media (120 h MB) were compared to differentiated myotubes (120 h MT). (D) Immunostaining images of MyHC (Red) and nuclei (DAPI, blue) on differentiated FSHD1 Cas9 and FSHD1 MAPK14/11-/- cells at 120 h time point with an image magnification at 60X. Scale bar: 100 μm. (E) Quantification of MHC + cells from figure B. MHC + and MHC − nuclei counts were aggregated from three separate imaging fields for each sample. The mean MI values ± Standard Error of the Mean (SEM) were calculated for three samples. Statistical significance was assessed using one-way ANOVA with Dunnett’s post-testing for multiple comparisons. The “ns” indicates that the observed differences were not statistically significant between unmodified and p38α/β double KO FSHD cells (p > 0.05). (F) Immunostaining images of MyHC (Red) and nuclei (DAPI, blue) on differentiated FSHD1 and FSHD2 cells that were treated losmapimod (_LOS) at 120 h time point. Scale bar: 100 μm. (G) Quantification of MHC + cells from figure F. MHC + and MHC − nuclei counts were aggregated from three separate imaging fields for each sample. The mean MI values ± Standard Error of the Mean (SEM) were calculated for three samples. Statistical significance was assessed using one-way ANOVA with Dunnett’s post-testing for multiple comparisons. The “ns” indicates that the observed differences were not statistically significant between untreated and losmapimod-treated FSHD cells (p > 0.05). (H) Relative DUX4 and DUX4 target RNA levels in FSHD1 and FSHD2 myocytes treated with vehicle (_Unmodified_Unmod) or 1 μm losmapimod (_LOS) during a 120-hour differentiation period. Myoblasts in growth media (120 h MB) were compared to differentiated myotubes (120 h MT). In all the qPCR data analysis RNA levels were normalized to housekeeping gene RPL30, followed by respective control groups (considered as 1) and knockout and pharmacological inhibition induced DUX4 and target gene expression fold changes to control were represented.