1. Volume 55, Issue 4, Pages 592-603 (August 2014)
Rbfox2-Coordinated Alternative Splicing of Mef2d and Rock2 Controls Myoblast Fusion during Myogenesis 
Ravi K. Singh, Zheng Xia, Christopher S. Bland, Auinash Kalsotra, Marissa A. Scavuzzo, Tomaz Curk, Jernej Ule, Wei Li, Thomas A. Cooper 
Molecular Cell 
Volume 55, Issue 4, Pages (August 2014)
DOI: /j.molcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2014 55, 592-603DOI: (10.1016/j.molcel.2014.06.035)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Rbfox2, but Not Rbfox1, Is Required for Terminal Skeletal Muscle Differentiation
(A) Neither early nor late myogenic markers are affected by Rbfox2 depletion during differentiation. Western blots of myogenic markers from C2C12 cultures treated with scrambled or Rbfox2 siRNAs and harvested at the indicated days following addition of differentiation medium (U is undifferentiated). Ponceau S (Pon S) staining of western blot membrane.
(B) Depletion of Rbfox2 but not Rbfox1 prevents myoblast fusion. Cultures were immunostained (day 4) for myosin heavy chain (Myh3) and DAPI counterstained after treatment with the indicated siRNAs, scrambled (Scr), Rbfox2 siRNA #1 (F2s1), Rbfox2 siRNA #2 (F2s2), Rbfox1 siRNA #1 (F1s1).
(C) Quantified fusion indices from (B). Standard error of the mean from three independent cultures is shown. ∗∗∗ indicates p ≤ as determined by one-way ANOVA using Newman-Keuls posttest.
(D) Western blot of parallel cultures from (B) for indicated proteins. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Rbfox2 Regulates Distinct Sets of Splicing Events in Proliferating Myoblasts and Differentiated Myotubes
(A) Venn diagrams showing overlap of genes that change in mRNA abundance (FDR <1%) (orange) and/or undergo alternative splicing (ΔPSI ≥20%) (green).
(B) RT-PCR validation of alternative splicing events using the same RNA sample used for RNA-seq.
(C) Venn diagram of differentiation-associated splicing changes and Rbfox2-sensitive splicing events in Undiff and Diff cultures.
(D and E) Line graphs of PSI for alternative splicing events affected by Rbfox2 knockdown (KD) primarily in Undiff cultures (D) or Diff cultures (E). Undiff and Diff cultures treated with scrambled or Rbfox2 siRNA are indicated by Undiff_Ctrl or Undiff_Rbfox2_KD and Diff_Ctrl or Diff_Rbfox2_KD, respectively. The splicing patterns for increased or decreased inclusion during differentiation are in the left and right panels, respectively.
(F) Ingenuity pathway analysis (IPA) analysis of Rbfox2-regulated events in differentiated cells. See also Figure S2 and Table S1, Table S2, and Table S4.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Identification of In Vivo Targets of Rbfox2 by iCLIP
(A) Enriched Rbfox motifs upstream and downstream (±150 bp) of Rbfox2-sensitive cassette exons were identified using MEME analysis. The blue and red lines indicate the distribution of the UGCAUG motif in all 375 Rbfox2-regulated cassette exons which decrease or increase, respectively, in Rbfox2 knockdown.
(B) Representative autoradiogram of Rbfox2-RNA complexes on [5′-32P]ATP-labeled RNA run on a polyacrylamide gel and blotted onto nitrocellulose filter. The red line indicates the position of the complexes isolated for library preparation.
(C) PCR-amplified iCLIP library in the indicated conditions run on a polyacrylamide gel and stained with ethidium bromide.
(D) Analysis for motif enrichment near the iCLIP crosslinked sites. The top fivve motifs in undifferentiated (Undiff) and differentiated (Diff) cultures are indicated with their comparative enrichment (Z score).
(E) Pie charts showing distribution of iCLIP tags in undifferentiated (Undiff) and differentiated (Diff) cultures.
(F) Distribution of Rbfox2 crosslinked tags are indicated near 375 Rbfox2 regulated exons (red), 4,040 alternative exons not regulated by Rbfox2 (blue), and 13,738 constitutive exons (black). See also Table S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Rbfox2 Is Required for Muscle-Specific Splicing of Mef2d
(A) RNA-seq data mapped to the mouse genome (mm9) on UCSC browser showing Mef2d reads from an undifferentiated (Undiff) culture treated with scrambled siRNA and 4 days differentiated (Diff) cultures treated with scrambled, Rbfox2, or Rbfox1 siRNAs.
(B) RT-PCR analysis of alternative splicing of the mutually exclusive alpha (left) and cassette beta (right) exons of Mef2d in C2C12 cells treated with scrambled (S) siRNA, Rbfox1 siRNA #1 (F1), or Rbfox2 siRNA #1 (F2) in Undiff and Diff cultures. Bottom panel indicates quantification of PSI in three independent replicates.
(C and D) Rbfox2 binding sites proximal to the Mef2d alpha (C) and beta (D) exons are required for regulation by Rbfox2. Top: Diagram of indicated Mef2d minigene, Mef2d genomic segment (brown) in a heterologous minigene (black). The positions of the primers used for RT-PCR are shown (blue arrows). Rbfox binding motifs within the vicinity of iCLIP crosslinks are indicated by red stars. The Rbfox motifs are 10 (GCATG) and 33 (TGCATG) nt downstream of α2 exon and 183 (GCATG) and 261 (TGCATG) nt upstream of the α1 exon. The β exon Rbfox motifs are 67 (GCATG), 184 (TGCATG), and 203 (TGCATG) nt downstream of the exon. Middle: Splicing assay of α exon (left) or β exon (right) minigenes containing wild-type (WT, GCATG, or TGCATG) or mutant (Mut, GACTG, or TGACTG) Rbfox2 binding sites following coexpression with Flag-tagged Rbfox2 in COSM6 cells. RT-PCR products separated on polyacrylamide gels, Flag western, and Ponceau S (pon S) staining of the membrane. Bottom: PSI quantification from three replicates. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
The Muscle-Specific Isoform of Mef2d Rescues the Fusion Defect Caused by Rbfox2 Knockdown
(A and B) Differentiated C2C12 cultures (day 4) expressing doxycycline-inducible (A) undifferentiated (Mef2dα1) or (B) differentiation-specific Mef2d (Mef2dα2β) isoforms using lentivirus. Cells were treated with scrambled (Scr) or Rbfox2 (F2) siRNAs (−) or (+) 1 μg/ml doxycycline and immunostained for Myh3 and myc-tagged Mef2d isoforms.
(C) Fusion index for experiments performed in (A) and (B). Asterisk indicates p ≤ 0.05, as determined by one-way ANOVA using Newman-Keuls posttest.
(D) Western blots using protein extracts from (A) and (B) assessing knockdown of Rbfox2 and expression of myc-Mef2d isoforms. Ponceau S (Pon S) staining shows equivalent loading.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Combinatorial Rescue of Fusion Defect in Rbfox2 Knockdown by Expression of Differentiation-Specific Isoform of Mef2d and Loss of Rock2
(A) Coimmunoprecipitation (coIP) of N-terminal Flag-tagged fragment of Rock2 using extracts from COSM6 cells expressing Flag-N terminus Rock2 fragment alone (N), or with myc-tagged C terminus fragments of Rock2 without (C0) or with one (C1) or two alternative exons (C2). Mock (M) lane indicates coIP using untransfected cells. Input and PonS staining indicates equivalent expression of Rock2 fragments.
(B) Differentiated C2C12 cultures (day 4) with lentivirus expressing the doxycycline-inducible, differentiation-specific Mef2d isoform. Cells were treated with scrambled (Scr) or Rbfox2 (F2) siRNAs (−) and (+) 1 μg/ml doxycycline (Dox) and immunostained for Myh3 and myc-tagged Mef2d isoforms.
(C) As in (B), except cells were treated with either scrambled plus Rock2 siRNA (R2) or Rbfox2 plus Rock2 siRNA (F2+R2).
(D) Western blots of experiments performed in (B) and (C) assessing knockdown of Rbfox2 or Rock2 and a late marker of differentiation myosin heavy chain 3 (Myh3). Ponceau S (Pon S) staining shows equivalent loading. See also Figure S4.
(E) Fusion index for experiments performed in (A) and (B); ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ as determined by one-way ANOVA using Newman-Keuls posttest.
Molecular Cell  , DOI: ( /j.molcel )
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