Extended Data Fig. 7: Chronic Bcl6 loss evokes compensatory signaling to maintain muscle mass.
From: Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis
(a) SUnSET analysis of puromycin incorporation into quadriceps (top) in Cre- tmx and Bcl6i-MKO mice 12 weeks after tamoxifen treatment. Puromycin labeling relative to total protein (Licor) by densitometry (bottom) (n = 3/group; P = 0.0017 by unpaired, two-tailed t-test with Welch correction). (b) Western blot and densitometry in quadriceps from Cre- tmx and Bcl6i-MKO males 15 weeks after tamoxifen treatment for LC3, 3 days after colchicine or vehicle treatment. (n = 3/group; Two-way ANOVA followed by Tukey’s multiple comparisons test was performed. P (Veh-Cre- tmx vs Col-Cre- tmx) = 0.0174; P (Veh-Cre- tmx vs Col-Bcl6i-MKO) = 0.0005; P (Veh-Bcl6i-MKO vs Col-Bcl6i-MKO) = 0.0083.) Flux is the difference in average LC3-II levels in colchicine minus vehicle (bottom right). (c) Western blot and densitometry of ubiquitin and total (Licor) protein in quadriceps from Cre- tmx and Bcl6i-MKO males 12 weeks after tamoxifen treatment (n = 4/group). (d) Quadrant plot of log2 fold change in gene expression in Bcl6i-MKO/Cre- tmx 12 weeks after tamoxifen treatment (chronic) versus Bcl6i-MKO/Cre- tmx 1 week after tamoxifen treatment (acute). (e) Ontology analysis of differential genes unique in chronic Bcl6 deletion. Cumulative hypergeometric statistical testing was used. (f) Log p-value versus Pearson correlation plotted for causal transcription factors identified by IMAGE that were unique to either acute or chronic Bcl6 deletion. Pink = high, green = medium, and gray = low confidence factors. Factors in acute dataset are shown as circles and factors in chronic are squares. (g) Causal transcription factors with opposing Pearson correlations to gene expression in acute versus chronic Bcl6 deletion. Acute = green circle, chronic = orange square. (h-l) Western blot and densitometry of (h) pSMAD1/5/8, Total SMAD1, and total (Licor) protein, (i) pFOXO1, Total FOXO1, and Actin, (j) p4EBP1, Non-phospho 4EBP1, Total 4EBP1, and Actin, (k) pAKT, Total AKT, and Actin and (l) pS6, Total S6, and Actin in quadriceps from Cre- tmx and Bcl6i-MKO males 12 week after tamoxifen treatment (n = 4/group). Unpaired, two-tailed t-test with Welch correction and all individual p-values can be found in the source data file. All bar graph data in (a-c, h-l) are represented as mean + SEM. * p < 0.05 and ** p < 0.01.
