Figure 3

HMGA1a over-expression prevents myogenic differentiation. (A) Immunofluorescence localization of myosin (red) in C2C12 (a-e) and in C2A1a cells (f-k) before (myoblast) and after induction (days 1 to 9). All bars represent 20 μm. Myotube formation was only observed in wild type C2C12 cells. In pictures a-e DNA staining by Hoechst is shown in blue, respectively. HMGA1a-eGFP is shown in green. (B) RT-PCR analysis to compare the expression of the myosin light chain (myosin lc) and α-actin in C2C12 cells (left) and in C2A1a cells (right) as described in Fig. 1. Expression of Hmga1, Gapdh and desmin are shown as controls. (C) RT-PCR to analyze expression profiles of marker genes for osteogenesis. Genes analyzed were alkaline phosphatase (AP) and osteocalcin. Gapdh expression is shown as control. HMGA1a over-expression did not affect AP and osteocalcin transcription. (D) Alkaline phosphatase activity as marker for osteogenesis of C2C12 and C2A1a cells on day 2 of differentiation (bright field images). AP activity was visualized using NBT/BCIP staining. Shown are overlays of bright field images and fluorescence images with corresponding DNA staining. Scale bar is 50 μm.