Palmer S , Groves N , Schindeler A , Yeoh T , Biben C , Wang CC , Sparrow DB , Barnett L , Jenkins NA , Copeland NG , Koentgen F , Mohun T , Harvey RP .

R03 AG14811 NIA NIH HHS , Wellcome Trust

	Figure 1. (A) DNA sequence of mouse Csl cDNA with predicted protein. (B) Alignment of human, mouse, rat, and Xenopus Csl proteins. Shaded amino acids indicate nonidentity. (Csl cDNA sequence data available from GenBank/EMBL/DDBJ under accession nos. AY026524 [mouse] and AF343894 [Xenopus]).
	Figure 2. Csl mRNA expression. (A) RNase protection analysis of Csl and control cyclophilin (Cyclo) mRNAs in adult mouse tissues. (B–K) Whole-mount in situ hybridization using Csl probe on mouse embryos. (B) Ventral view of E8.25 embryo. (C) Section of the embryo shown in B. Arrows indicate expression on the ventral surface of the fusing heart tube. (D) Ventral view of E8.5 embryo. (E) Section of embryo depicted in D showing Csl expression at the outer curvature of the ventricles. (F–H) E10.5 embryo viewed from the ventral and left and right sides, respectively. Arrow in F indicates rib of expression in outflow tract. Arrow in G indicates expression in presumptive left atrial appendage. (I) Section of E10.5 heart. Note that expression is restricted to the outer curvature of the ventricles and atrial appendages. (J) E9.5 Nkx2-5−/− embryos (−/−) compared with wild-type sibling (+/+). (K) E13.5 embryos comparing expression of Csl and myogenin. as, atrial septum; at, atrium; avc, atrioventricular canal; lv, left ventricle; oft, outflow tract; rs, right sinus horn; rv, right ventricle; tb, trabeculae.
	Figure 4. Csl protein expression. (A) Western blot analysis of Csl expression in adult tissues. An equal mass of protein was loaded in each lane. “Blocked” indicates lanes in which Csl antibody was preabsorbed with excess recombinant Csl protein. (B) Western blot analysis of Csl expression in postnatal day (P)1, P8, and P21, and/or adult tissues from wild-type and male CslAPΔ knockout mice using Akt levels as loading control. (C) Comparison of levels of Csl expression in heart and different skeletal muscles using Hsp 70 levels as control. Note the additional Hsp 70 isoform enriched in soleus. (D–F) Immunofluorescence analysis of Csl expression in C2C12 cells after 1, 2, and 4 d, respectively, in differentiation medium. (G) Immunofluorescence on 4-d culture after blocking as in A. bm, bipolar mononuclear myocytes; Diaphm, diaphragm; Gastroc, gastrocnemius; rm, rounded mononuclear myocytes. Bar, 100 μm.
	Figure 3. (A) Csl-targeting construct relative to wild-type and targeted alleles. Large arrowheads indicate direction of LoxP sites. Black shading within exon 2 indicates Csl-coding region. Arrows indicate PCR genotyping primers. hPAP, hPAP gene; pgk-Neo, neomycin resistance gene cassette. (B) Example of multiplex PCR genotyping of targeted (582 bp), wild-type (517 bp), and Cre-deleted (321 bp) alleles. M, MluI; P, PvuII; X, XbaI.
	Figure 5. Tissue distribution of Csl protein. Sections from newborn (A–D) and adult (E–I) mice were analyzed for Csl and/or myosin heavy chains using immunofluorescence (A, B, D–F, H, and I) or for Csl by staining for hPAP (C and G). (A) Section showing expression in heart and adjacent skeletal muscles. (B) Section from a CslAPΔ mouse. (C) Section from CslAPΔ mouse stained for hPAP. Arrowheads show nonspecific staining. (D) Section showing Csl expression in smooth muscle of the stomach. (E and F) Near adjacent sections showing muscle expression of Csl (E) and fast myosin heavy chain IIb (F). (G) Fiber-type pattern of Csl expression in CslAPΔ mouse revealed by hPAP staining. (H and I) Double immunofluorescence showing muscle fiber–type pattern of Csl (H) and slow myosin heavy chain I (I). Arrowheads indicate slow fibers showing the highest Csl fluorescence. Bars indicate length in micrometers. at, atrium; bl, bladder; dm, diaphragm; gs, gastrocnemius; ic, intercostal muscle; li, liver; pl, plantaris; si, small intestine; so, soleus; st, stomach; ve, ventricle. Bars: (A, B, and D) 500 μm; (C) 1 μm; (E–G) 200 μm; (H and I) 100 μm.
	Figure 6. Subcellular localization of Csl protein. (A and B) Double immunofluorescence on a section of E18.5 skeletal muscle showing localization of Csl (A) and α-actinin (B). (C) High power double immunofluorescence confocal image (overlay) of a section of stretched adult soleus (see D–F) showing Csl (green) and α-actinin (red). Arrowheads indicate weak Csl staining over the M-line. (D–F) Double immunofluorescence confocal image of a section of stretched adult soleus showing Csl (D), α-actinin (E), and overlay (F). (G–I) Confocal images of Csl (G) and talin (H) with overlay (I). (J) Overlay of Csl and talin double immunofluorescence in myotendenous junctions (arrowheads). (K and L) Transverse sections of E17.5 myotubes showing Csl (K) and α-actinin (L). (M–O) Double immunofluorescence of Csl (M) and α-actinin (N) with overlay (O) in adult ventricular myocytes. Arrowheads indicate Csl staining over the M-line. mf, myofiber; tn, tendon. Bars: (A, B, D–I, and K–L) 20 μm; (C) 10 μm; (J) 50 μm.
	Figure 7. Effect of constitutive Csl expression in C2C12 myoblasts. (A and B) Phase–contrast images of stable C2C12 clones constitutively expressing GFP (A) or myc-Csl (B). (C) Mean myoblast area (± SEM) of parental C2C12 cells and a series of transfected myc- or FLAG-Csl–expressing clones. GV is transfected with GFP vector only; ME1/2 are lines expressing myc-Csl driven by EF1α promoter; MM1/2 are lines expressing myc-Csl driven by differentiation-dependent MLC1 promoter/enhancer. Only ME1, ME2, and FC18 express Csl in myoblasts. (D) Overlay of forward light scatter histograms from a single experiment in which C2C12 stable clones (GV, ME1, ME2, MM1, MM2) and parental cell line were compared. As positive control, GV cells were resuspended in hypotonic 0.5× PBS causing a rightward shift. (E) Mean forward light scatter (± SEM) determined from three separate experiments and plotted as a percentage of controls. (F) Mean percentage (± SEM) of closure of 150-μm wounds in confluent monolayers after 6 h. (G) Anti-myc antibody immunofluorescence in a myc-Csl–expressing myoblast. (H) GFP fluorescence in a C2C12 myoblast transiently expressing GFP–Csl fusion protein. Bars, 20 μm.
	Figure 8. Effects of constitutive FLAG-Csl expression in differentiating C2C12 myogenic cells. Phase–contrast and immunofluorescence images of control (F5; left panels) and FLAG-Csl–expressing (FC18; right panels) C2C12 cells after 3 d of differentiation. (A and B) No treatment. (C and D) With 100 ng/ml IGF-1. (E and F) With 100 ng/ml IGF-1 plus 1 μM CsA. (G and H) MF20 immunofluorescence on cells treated with IGF-1. Bars, 100 μm.
	Figure 9. Myogenic gene expression and protein to DNA ratios in control (F5) and FLAG-Csl (FC18) C2C12 cells in the presence and absence of IGF-1 (100 ng/ml). (A) Northern blot analysis of total RNA from cells cultured for 0–5 d in differentiation medium using indicated probes. Ethidium bromide (EtBr) staining of 28S and 18S ribosomal RNAs serves as loading control. Two mRNA species are produced from the transgene due to differential 3′ untranlated region processing. (B) Total protein/total DNA ratios ± SEM at different times of differentiation. Myog, myogenin; Mhc, embryonic myosin heavy chain.
	Figure 10. NFAT, MEF2, and calcineurin activity in control (F5) and FLAG-Csl (FC18) cells with and without IGF-1 (100 ng/ml). (A and B) Mean NFAT-dependent transcriptional activity (± SEM) measured using a transfected luciferase reporter gene driven by the myoglobin promoter (Mygb), compared with the same promoter with mutated NFAT-binding sites (Mygb Δ-NFAT). (C and D) Mean MEF2-dependent transcriptional activity (± SEM) measured using a transfected luciferase reporter containing three MEF2-binding sites (3×MEF) compared with the minimal vector (Vector only). (E) Levels of calcineurin phosphatase activity measured in vitro as picomoles of 32P-radiolabeled phosphate released per minute per microgram of extract (pmol/min/μg). (F) Effect of CsA (1 μM) on NFAT activity measured using the wild-type myoglobin (Mygb) reporter at day 4 in FC18 cells after treatment with IGF-1 (left) or after transient transfection of activated calcineurin A subunit (Act-CnA) (right).

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