CLP-1 associates with HDACs at the onset of C2C12 cell differentiation. (A) Immunoprecipitation (IP) of C2C12 cell lysates from growth (G) and differentiation medium at 24 hours (D1) and at 72 hours (D3) with antibodies specific for HDAC1, HDAC3 and HDAC5, and western blot with anti-CLP-1 and anti-cdk9 antibodies. IP with antibody alone served as a control for immunoreactivity (Ab). Western blot with anti-CLP-1 and anti-GAPDH antibodies served as input controls. Data represent one of three separate experiments for each antibody. Bottom right panel: western blot of total cell lysates with anti-HDAC1, anti-HDAC3 and anti-HDAC5 antibodies. Western blot with GAPDH served as input control. (B) Left panel: nuclear (Nuc) and cytoplasmic (Cyto) fractions of C2C12 cells in differentiation medium at 24 hours were subjected to direct western blotting with antibodies specific for CLP-1, cyclin T1, MyoD and GAPDH. Right panel: lysates were subjected to IP with anti-HDAC5 antibody and western blotting with anti-CLP-1 antibody. Direct western blot of CLP-1 served as an input control.

C2C12 CLP-1 +/− cells are differentiation deficient. (A) PCR using DNA from C2C12 CLP-1 +/+ cells and one CLP-1 +/− clone. Primers generate a 457 base pairs product for the CLP-1 gene and a 383 base pairs product for the mutated allele. M denotes DNA size ladder. Data are shown for one representative clone. (B) Western blot of C2C12 CLP-1 +/+ cells and C2C12 CLP-1 +/− cells in growth medium probed with anti-CLP-1 antibody. GAPDH served as a loading control. Western blot is shown for one representative clone. The analysis was performed on three independently isolated C2C12 CLP-1 +/− cell clones, and means are depicted graphically + s.e.m.; GAPDH was used for normalization (*P<0.05). (C) Western blot of lysates from C2C12 CLP-1 +/+ and +/− cell cultures in growth medium (G) and differentiation medium at 24 hours (D1) and at 72 hours (D3), probed with anti-CLP-1, anti-myogenin, anti-PCNA, anti-cyclin D1 and anti-MyoD antibodies. GAPDH served as a loading control. The analysis was performed three times, and means are depicted graphically + s.e.m.; GAPDH was used for normalization (*P<0.05, **P<0.01). (D) Immunofluorescence of C2C12 CLP-1 +/+ and C2C12 CLP-1 +/− cells in growth and differentiation medium using actin stain (green) and co-stained with anti-CLP-1 (red) or DAPI nuclear stain (blue). This analysis was performed on three independently isolated C2C12 CLP-1 +/− cell cultures. (E) Graphical analysis of immunofluorescence of C2C12 CLP-1 +/+ (WT) and +/− cells in growth media (GM) and differentiation media (DM) for 0, 24, 48 and 72 hours. Antibodies to PCNA and myosin heavy chain were used, and DAPI was used as a nuclear stain. At each time point, cells were counted (n=500) and the ratio of cells expressing PCNA or myosin heavy chain versus total cells is depicted graphically (n=3).

Association of CLP-1 with cdk9 and MyoD is dynamic in C2C12 cell differentiation. Whole-cell lysates were collected from growth medium (G) and differentiation medium at 24 hours (D1) and at 72 hours (D3). (A) Equal amounts of protein were subjected to SDS-PAGE followed by immunoblotting. Myogenin (MyoG) served as a marker of differentiation. GAPDH served as a loading control. (B) Immunoprecipitation (IP) of C2C12 cell lysates with anti-cdk9 antibody, and western blotting with anti-CLP-1 antibody. Western blot with anti-cdk9 antibody served as a control of total immunoprecipitated protein. (C) IP of C2C12 cells with anti-MyoD antibody and western blotting with anti-CLP-1 antibody. Western blot with anti-MyoD antibody served as a control of total immunoprecipitated protein. (D) IP using C2C12 cell lysates with anti-MyoD antibody and western blotting with anti-HDAC1 and anti-PCAF antibodies. Direct western blots of HDAC1 and PCAF served as input controls. In all cases, IP with antibody alone served as a control for immunoreactivity (Ab). Data shown represent one of three separate experiments.

Analysis of the cyclin D1 promoter in C2C12 cell differentiation. (A) Analysis of C2C12 cells in growth medium (G), and differentiation medium at 24 hours (D1) and 72 hours (D3) by western blotting with antibodies specific for CLP-1, myogenin and cyclin D1. GAPDH served as a loading control. (B) ChIP was performed on C2C12 cells using antibodies specific for MyoD, CLP-1, HDAC1, HDAC3 and HDAC5. Nonspecific IgG and anti-actin antibody served as negative controls. Precipitated DNA was amplified by PCR for regions of the cyclin D1 gene corresponding to the 5′ upstream promoter region encompassing two E-box sequences. Input DNA represents 10% of total chromatin used in each reaction. Primers specific to GAPDH were used before (input) and after immunoprecipitation as a control to monitor immunoprecipitation specificity. Data represent one of three separate experiments. (C) Luciferase assay in C2C12 cells in growth media (GM) and for 24 hours in differentiation media (DM). Cells were co-transfected with cyclin D1-luciferase plasmid along with expression plasmids encoding MyoD, CLP-1 and HDAC5. Renilla luciferase expression was used for normalization. Luciferase activities for cyclin-D1 in differentiation media is expressed relative to the mean value derived from cells co-transfected with cyclin D1-luciferase and Renilla luciferase in growth media. Luciferase activity for + MyoD, + MyoD + CLP-1 and + MyoD + CLP-1, + HDAC5 in differentiation media are expressed relative to the mean value derived from cells co-transfected with cyclin D1-luciferase and Renilla luciferase in differentiation media. Experiments were repeated three times. Bars show mean + s.e.m., *P<0.05.

Analysis of expression of P-TEFb and MRFs in mouse skeletal muscle. (A) Immunoprecipitation (IP) of skeletal muscle lysates from wild-type E12 embryo and adult, with anti-cdk9 and anti-MyoD antibodies and western blotting with anti-CLP-1 antibody. IP with antibody alone served as a control for immunoreactivity. Western blot of total protein served as an input control. GAPDH served as a loading control. (B) Western blotting of skeletal muscle tissue lysates from E12 mouse embryos wild-type (WT), heterozygote (+/−) and homozygote (−/−) for the CLP-1 allele with anti-CLP-1, anti-CLP-2, anti-cdk9, anti-cyclin T1 and anti-RNA polymerase II (Pol II) antibodies. GAPDH served as a loading control. (C) Western blotting of skeletal muscle tissue lysates with anti-MyoD, anti-myogenin and anti-myosin heavy chain (MHC) antibodies. GAPDH served as a loading control. Data represent one of three separate experiments with one embryo per experiment. Relative MyoD, myogenin and MHC levels are depicted graphically as means + s.e.m. GAPDH was used for normalization (*P<0.05). (D) Immunohistochemical analysis of coronal section of CLP-1 +/+ and CLP-1 −/− E12 mice. Sections were stained with DAPI (blue), anti-CLP-1 antibody (red) and anti-myogenin antibody (green). Merge of CLP-1 and myogenin is depicted in the fourth panel (yellow). In diagram above, NT denotes location of the neural tube, and red square denotes relative localization of section. Images were captured at 10× magnification.