ECM produced by BMP-2 treated myoblasts induces alkaline phosphatase in C2C12 cells. A. C2C12 cells were induced to differentiate for 6 days. Unpermeabilized cells (E-G) or coverslips after cell removal by EDTA treatment (I-K) were stained with anti-fibronectin (FN), anti-laminin (LN) or anti-perlecan (PER) antibodies. H and L show anti-tubulin (TUB) staining after permeabilization. FITC-conjugated secondary antibodies were used. Phase contrast microscopy is shown (A-D). Bar = 25 μm. B. C2C12 cells were induced to differentiate for 6 days in the absence (MTs) or presence of 5 nM BMP-2 (BMP-2). Unpermeabilized cells (A, D) or ECM (G, J) obtained as described in A were stained with anti-fibronectin antibodies. FITC-conjugated secondary antibodies were used. Nuclear staining was performed with 1 μg/ml Hoechst 33258 (B, E, H, K). Phase contrast microscopy for cells is shown (C and F) and staining without the primary antibody is shown in I and L. Bar = 25 μm. C. C2C12 cells were plated on ECM from myotubes (ECM MTs) or BMP-2 treated cells (ECM BMP-2), as described above, and induced to differentiate in the absence or presence of 1 μg/ml soluble BMP receptor IA (BMPR-IAsoluble). ALP activity was determined at day 6. Data are presented as mean ± S.E.M. of three independent experiments performed in triplicate. * = significantly different from the other values, but not between them, p < 0.015 (ANOVA followed by Tukey-Kramer multiple comparisons test).