Figure 4

HMGA1a over-expression interferes with chromatin organization during myogenesis. (A) Depletion of HMGA1a-eGFP after HMGA1a-siRNA treatment is visual through the loss of eGFP fluorescence in C2A1a cells (b). Mock transfected C2A1a cells are shown in a. The corresponding Hoechst stained images are shown in a' and b'. The bar represents 20 μm. (B) Western blot analysis showed considerable depletion of endogenous and eGFP-tagged HMGA1a proteins, respectively, 12-24 hours after siRNA treatment of C2C12 and C2A1a cells. Ponceau staining (P) of the core histones is presented as loading control. Protein molecular mass is indicated in kDa. (C) Heterochromatin foci (chromocenters) in C2C12 and C2A1a cells were quantified (n = cell number) and plotted as indicated. (a) In myoblasts (day 0) the number of chromocenters is identical in both cell lines. (b) During terminal muscle differentiation of the C2C12 cells, the number of heterochromatin foci decreases due to chromocenter clustering (grey columns) whereas the chromocenter number in C2A1a cells remains comparable to the number in myoblasts (green columns). (D) C2A1a control cells (a) and C2A1a cells 12-24 hours after siRNA treatment (b). HMGA1 depletion, indicated by absence of HMGA1a-eGFP, results in a higher number and a reduced size of chromocenters (arrowheads) (b'). The bar represents 10 μm. (E) Depletion of HMGA1 by siRNA in C2A1a myoblasts increased the chromocenter number (red columns) compared to C2A1a control cells (green columns). A similar chromocenter dissociation was detected in C2C12 wild type cells on differentiation day 3 (grey columns) prior to fusion during terminal differentiation.