1. Volume 21, Issue 11, Pages 3003-3011 (December 2017)
ATP Citrate Lyase Regulates Myofiber Differentiation and Increases Regeneration by Altering Histone Acetylation 
Suman Das, Frederic Morvan, Giulio Morozzi, Benjamin Jourde, Giulia C. Minetti, Peter Kahle, Helene Rivet, Pascale Brebbia, Gauthier Toussaint, David J. Glass, Mara Fornaro 
Cell Reports 
Volume 21, Issue 11, Pages (December 2017)
DOI: /j.celrep
Copyright © 2017 The Author(s) Terms and Conditions

2. Cell Reports 2017 21, 3003-3011DOI: (10.1016/j.celrep.2017.11.038)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 ACL Is Required for Skeletal Muscle Differentiation In Vitro
ACL was downregulated by siRNA (40 nM) in confluent human primary myoblasts, and cells were analyzed at day 6 (A and C) or during the differentiation process (B).
(A) Myotubes were fixed and stained for fast and slow MyHC along with DAPI. Graphs represent means ± SEM (n = 9, 3 independent experiments; myotube number, fiber width, and total nuclei, Mann-Whitney test; differentiation index, Wilcoxon signed-rank test).
(B) Protein and mRNA abundances were analyzed at the indicated time points by immunoblotting and by qRT-PCR, respectively.
(C) Following knockdown of ACL, myoblasts were treated with 30 nM IGF-1 in differentiation medium. (B and C) Representative blots are shown (3 independent experiments; one-way ANOVA). Quantification of immunoreactive bands from blots was performed by densitometric analysis and data are mean intensity ± SEM; GAPDH was used as the loading control; qRT-PCR data are shown as mean fold increase ± SEM of siCtrl at day 2.
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4. Figure 2 ACL Regulates the Differentiation of SCs
Mouse SCs were infected with adACL or adenovirus empty vector (adEV) as control (A–C and E); in parallel, some were transfected with siACL or with non-targeted siCtrl (D).
(A) Cell proliferation was analyzed by BrdU staining after 24 or 36 hr, and graph represents average ± SEM percentage of BrdU-positive cells (6 mice/group).
(B) At 48 hr after infection, the expression of Acl and muscle-specific genes was analyzed by qRT-PCR. Graphs represent mean fold increase ± SEM of control adEV (4 mice/group).
(C) Fusion index, fiber area, and fiber width were analyzed in SCs differentiated for 48 hr, as described in the Experimental Procedures. Graphs represent mean ± SEM (3 mice/group).
(D) mRNA expression of Acl, Myh1, Myh2, Myh4, Myh7, and Myod was quantified by qRT-PCR. Graphs represent mean fold ± SEM of siCtrl (5 mice/group).
(E) Expression of Myod, Myog, and Mef2C was analyzed in differentiating SCs by qRT-PCR. Graph represents mean fold increase ± SEM of control adEV (4 mice/group; Mann-Whitney test except for D, Wilcoxon signed-rank test).
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5. Figure 3 ACL Promotes Histone Acetylation at the MyoD Locus via p300
ACL was downregulated by siACL in human primary myoblasts (A and D) and C2C12 (B and C), and cells were analyzed after 2 days in differentiation medium.
(A) Acetylated histones and total H3 levels were analyzed by immunoblotting. Representative blots and densitometric analysis (intensity ± SEM) are shown.
(B) Acl and Myod expression was analyzed by qPCR. Graphs show mean fold increase ± SEM of siCtrl.
(C) ChIP analysis was performed using antibody to acetylated H3(K9/K14) or control antibody.
(D) Following knockdown of ACL, myoblasts were treated with 30 nM IGF-1, and they were analyzed for the enrichment of Ac-H3(K9/K14) or Ac-H3(K27) at the MyoD promoter by ChIP.
(E) ACL was overexpressed by adACL with adEV as control in human primary myoblasts, and enrichment of Ac-H3(K9/14)/Ac-H3(K27) at the MYOD promoter was analyzed by ChIP during differentiation.
(F and G) C2C12 myoblasts were infected with adACL or adEV, and they were treated in the absence or presence of 10 μM C646 in differentiation medium for 2 days. (F) Myod mRNA expression was analyzed by qRT-PCR. Graph shows mean fold increase ± SEM of vehicle control. (G) Same as in (C) is shown.
(C–E and G) Data are presented as percentage enrichment ± SEM versus input chromatin DNA (A, C, and E–G, 4 independent experiments; B and D, 3 independent experiments; A, D, and E, two-way ANOVA; C, one-way ANOVA; B, Student’s t test; F and G, Mann-Whitney test).
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6. Figure 4
ACL Overexpression Improves Muscle Regeneration after Cardiotoxin-Induced Injury
AAVs expressing ACL (AAV-ACL) or GFP (AAV-GFP) were injected into the tibialis muscle.
(A) ACL expression was analyzed at the indicated days post-cardiotoxin injection by qRT-PCR. Graph represents mean fold increase of AAV-GFP (8 mice/group).
(B) Tibialis weight was normalized by initial body weight (IBW) and computed as percentage of control of non-injected muscle at day 21 after cardiotoxin injection (10 mice/group).
(C and D) Tissue sections from AAV-ACL- or AAV-GFP-infected tibialis muscles at day 21 post-cardiotoxin injection were stained with anti-laminin antibody; the number of fibers (C) and average fiber cross-sectional area (CSA) (D, inset) were analyzed.
(D) Frequency histograms show the distribution of myofiber CSA (6 mice/group).
(E–G and I) qRT-PCR analysis of the abundance of transcripts involved in the regeneration process in tibialis muscles at days 3, 7, 14, and 21 post-cardiotoxin injection (6–8 mice/group). (E) Pax7, Myf5, Myh3 and Myh8; (F) Myog; (G) Myod; and (I) Myh1, Myh2, Myh4 and Myh7.
(H) Enrichment of acetylated H3-K9/14 at the Myod promoter (CER, DRR, and PRR) by ChIP was analyzed at day 3 after cardiotoxin-induced injury (6 mice/group).
(J) Summary scheme of the described findings.
(A–I) Graphs represent mean ± SEM or ± SD (C and H; A, multiple t test with Holm-Sidak method; B–D and H, Mann-Whitney test; E–G and I, two-way ANOVA).
Cell Reports  , DOI: ( /j.celrep )
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