Figure 2

Characterization of the HMGA1a-eGFP over-expressing cell line C2A1a. (A) Sustained expression of HMGA1a-eGFP in C2A1a cells throughout myogenic induction analyzed by RT-PCR and Western blotting (WB) as indicated. Both, fusion protein (~45 kDa) and endogenous protein (~17 kDa) were detected using an HMGA1-specific antibody. Note the sustained expression throughout myogenic induction. Loading controls are as mentioned in Fig. 1. (B) Localization of HMGA1a-eGFP in living C2A1a cells. Note the concentrated localization in heterochromatin foci (chromocenters) during interphase (a) and on pericentromeric regions during mitosis (b). Scale bars represent 10 μm. (C) Immunolocalization on fixed C2A1a cells using HP1α-specific antibodies. Note the colocalization of HMGA1a-eGFP (a') and HP1α (a'') in the chromocenters of C2A1a cells. DNA was stained with Hoechst (a). An overlay of a-a'' is shown in a'''. The bar represents 10 μm. (D) HMGA1a-eGFP (green) colocalizes with the H3K9me3 and H4K20me3 specific immunolocalizations (red) in C2A1a cells. DNA was stained with Hoechst. The bar represents 10 μm. (E) Cell cycle phases are unaffected in the C2A1a cell line. DNA from C2C12 and C2A1a was stained with propidium iodide and 20,000 cells from each cell line were analyzed by FACS. Cell numbers (counts) are plotted against the relative DNA content of the cells. Phase distribution was analyzed with modfit Lt3.1. 56.14% of C2C12 cells were in G1 phase, 29.54% in S phase, and 14.33% in G2 phase. In C2A1a cells 60.95% of the cells were in G1 phase, 24.43% in S phase, and 14.63% in G2 phase.