Extended Data Fig. 6: Loss of Bcl6 reduces the rate of muscle protein synthesis.

(a) Heatmap of ribosome protected fragment (RPF) sizes in sequencing libraries of tamoxifen-treated Bcl6^i-MKOmRiboTag and mRiboTag mice. (b) Pie chart distribution of mapped RPF reads. (c) Empirical cumulative distribution frequency (Ecdf) plots of the translation efficiencies (natural logarithm of TE) in quadriceps for all genes. Bcl6^i-MKO mRiboTag vs mRiboTag P = 2.2e-16 by two-sided Kolmogorov-Smirnov testing (d) Metagene distribution plot showing normalized ribo-seq RPF reads from the TSS to TTS for all genes (left) and genes with increased TE (right). (e) Western blot and densitometry of ubiquitin protein and actin in quadriceps from Cre^- tmx and Bcl6^i-MKO males 1.5 weeks after tamoxifen treatment (n = 4/group). (f) EIF4E co-immunoprecipitation and western blots for EIF4G (top) and 4EBP1 (bottom) in gastrocnemius from Cre^- tmx and Bcl6^i-MKO males 1 week after tamoxifen treatment (n = 2/group). (g) Western blot and densitometry of p4EBP1, Non-phospho 4EBP1, Total 4EBP1, and total (Memcode) protein in quadriceps from 10 week old Bcl6^fl/fl and Bcl6^MKO males (n = 4/group). P(Bcl6^fl/fl vs Bcl6^MKO) for p4EBP1 = 0.0009, total 4EBP1 = 0.0004, and nonP 4EBP1 = 0.0002 by unpaired, two-tailed t-test with Welch correction. (h) qPCR of protein synthesis and degradation regulators in n = 4 Cre⁺ oil, n = 4 Cre^- tmx and n = 5 Bcl6^i-MKO male mice 7 days after treatment. One-way ANOVA with Dunnett’s multiple comparisons test showed that P(Cre⁺ oil vs Bcl6^i-MKO) for Eif4ebp1 = 0.0086, Mstn = 0.0018, Igf1 = 0.0332 and Ar = 0.0420; P(Cre^- tmx vs Bcl6^i-MKO) for Eif4ebp1 = 0.0163, Mstn = 0.0011, Igf1 = 0.0140 and Ar = 0.0330. (i, j) Analysis of Eif4ebp1 knockdown efficiency in muscles (n = 3 mice/group for scramble and Eif4ebp1 shRNA1 and n = 2 mice/group for Eif4ebp1 shRNA2). (i) qPCR of Eif4ebp1 and (j) western blot and densitometry of 4EBP1 in Cre^- tmx and Bcl6^i-MKO male mice transduced with scramble (black border), Eif4ebp1 shRNA1 (green border), or Eif4ebp1 shRNA2 (yellow border) and treated with tamoxifen one week after viral infection. Tissues were analyzed eight weeks later. All bar graph data in (e, g-j) are represented as mean + SEM. # p < 0.05, ## p < 0.01 for Cre⁺oil vs Bcl6^i-MKO and * p < 0.05, ** p < 0.01, *** p < 0.001 for Cre^-tmx vs Bcl6^i-MKO and Bcl6^fl/fl vs Bcl6^MKO.

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