Figure 5
HMGA1a over-expression alters the chromatin composition. (A) Western blot analyses comparing the expression of HMGB1, HMGN1, and histone H1 in C2C12 cells and C2A1a cells in myoblasts (day 0) and on days 1 and 3 of differentiation. The antibody used [43] recognizes all histone H1 variants. Proteins of 1.5 × 105 cells were analyzed in 15% SDS-PAGE. Lamin A/C expression and Ponceau S staining are shown as loading controls. (B) Histone H1 levels are unaffected in C2C12 cells after HMGA1 knock-down. Western blot was as described in Fig. 5A. Antibodies used are indicated. Histone H1 antibody used was directed against all H1-sub-variants (Abcam). Cells were untreated (-), siRNA treated (+) or transfected using siRNA control oligos (Ctrl). Ponceau S staining is shown as loading control. (C) Western blot analysis comparing MeCP2 expression in C2C12 and C2A1a cells. Note the premature MeCP2 expression in C2A1a cells. (D) Immunofluorescence localization of MeCP2 in C2C12 and C2A1a cells. MeCP2 is hardly detectable in C2C12 myoblasts (a') but accumulates in chromocenters of C2A1a cells (b). MeCP2 is concentrated in fused chromocenters in C2C12 cells (c') and is accumulated in chromocenters of C2A1a cells on day 6 after induction (d'). Note the absence of chromocenter clustering. Corresponding DNA staining is shown in a, b, c and d, respectively. Scale bars represent 10 μm.