Fig. 4: Reduction of VPS39 levels alters the epigenome and insulin signaling in human myoblasts.

a–c Specific phosphorylation (intensity of phosphorylation divided by total levels for each corresponding protein) for Akt at Ser473 (a, left panel) and Thr308 (a, right panel), TBC1D4 at Thr642 (b), GSK3-α at Ser21 (c, left panel) and GSK3-β at Ser9 (c, right panel) in the basal state and after insulin stimulation (100 nM, 30 min) in VPS39-silenced myoblasts (siVPS39, purple bars) and negative control (NC, gray bars) at day 3 of differentiation. n = 4 independent experiments. NC in the basal state is set to 1. Representative blots are shown. #p < 0.05, ##p < 0.01 for siVPS39 vs. NC for each treatment, and *p < 0.05, **p < 0.01, ***p < 0.001 for basal vs. insulin (Fisher’s LSD test). For exact p-values see Supplementary Table 1. d Kinetics of histone acetyltransferase (HAT) activity measurement (left panel). HAT (area under the curve) and histone deacetylase (HDAC) (fixed point) activity in nuclear extracts from siVPS39 (purple) and NC (gray) myoblasts at day 3 of differentiation (right panel). n = 4 independent experiments. NC is set to 1. *p < 0.05 for siVPS39 vs. NC. p = 0.0126 (HAT). e Protein levels (Western blot) of epigenetic enzymes from siVPS39 (purple bars) and NC (gray bars) myoblasts at day 3 of differentiation. Protein levels were measured in whole-cell lysates, or in nuclear and cytosolic fractions as indicated. n = 6 (DNMT1, DNMT3A, HDAC5 [nucleus and cytoplasm]), n = 3 (DNMT3B), n = 5 (EZH2, HAT1 [nucleus], p300 [nucleus and cytoplasm], HDAC4 [nucleus]), n = 4 (HAT1 [cytoplasm]), n = 8 (HDAC4 [cytoplasm]). NC is set to 1. Representative blots are shown. Statistical analysis was performed on log2-transformed values. *p < 0.05, **p < 0.01 for siVPS39 vs. NC. p = 0.0259 (DNMT1), p = 0.0374 (DNMT3B), p = 0.0081 (EZH2), p = 0.0697 (HAT1 [nucleus]), p = 0.0309 (p300 [nucleus]), p = 0.0493 (HDAC4 [nucleus]), p = 0.001 (HDAC5 [nucleus]), p = 0.0463 (HDAC5 [cytoplasm]). DNMT, DNA methyltransferase, HAT histone acetyltransferase, HDAC histone deacetylase. f The number of genes with altered DNA methylation at one or more CpG site (dark gray) or no change (light gray) among the 2635 genes with differential expression in siVPS39 vs. NC myoblasts. g The number of observed (black bars) and expected (white bars) genes for a selection of GO cellular processes enriched among genes with differential DNA methylation and gene expression in siVPS39 vs. NC myoblasts. Bars sorted by ratio (observed/expected). GO gene ontology. h–i Western blot analysis of acetylated histone 3 (ac-H3) levels related to the total amount of H3 in siVPS39 (purple bars) and NC (gray bars) myoblasts at day 3 of differentiation (h), and at days 0, 3, and 7 of differentiation (i). n = 5 independent experiments. NC/WT is set to 1. Representative blots are shown. *p < 0.05 for siVPS39 vs. NC in (h). p = 0.012. #q < 0.05, ####q < 0.0001 for comparisons between time points within each genotype, and *q < 0.05, ***q < 0.001 for siVPS39 vs. NC at each time point in (i). For exact q-values see Supplementary Table 1. Diff. differentiation. j–k Apoptosis measured as Caspase 3/7 activity (j, n = 3 independent experiments) and nucleus size (area of the DAPI-stain measured in the HCS assay) (k, n = 8 independent experiments) in siVPS39 (purple bars) and NC (gray bars) at day 3 of differentiation. NC is set to 1. *p < 0.05, ****p < 0.0001 for siVPS39 vs. NC. p = 0.0259 (j), p = 0.000051 (k). Bars (in (a–c, d), right panel, and (e, h–k)) or points (in (d), left panel) represent mean values and error bars display SEM (a–e, h–k). The effects of genotype, and insulin-treatment or differentiation stated above the graphs were calculated with repeated measures two-way ANOVA (a–c, i). P-values were adjusted for multiple comparisons with false discovery rate (FDR) analysis (g, i). Statistical significance determined by paired two-tailed t-test (d–e, h, j–k).