Fig. 3.

Endogenous BMP production and BMP activity during ESCs differentiation in CDMM: a RT-PCR mRNA quantification (ratio over β-Actin) of BMP2 and BMP4 in expanding ESCs (undifferentiated), ESCs at the second and fourth day of step II, expanding RAW 264.7 cell line (RAW) and mouse mesenchymal stromal cells (MSCs, passage 3). b Secreted BMP2 quantification by ELISA in cells as in (a). c–e Ntub (green) and phospho-SMAD1/5/8 immunodetection (nuclear red staining over DAPI nuclear counterstaining) in ESCs at step II (day 2) in CDMM (c), 5 h after the addition of 400 nM Noggin to CDMM (d) and 5 h after the addition of 50 ng/ml BMP4 to CDMM (e). f Phospho-SMAD1/5/8 immunodetection (red staining over DAPI) in undifferentiated ESCs. Scale bars 30 μm. g, h Pixel intensity distribution (fraction of nuclei with given pixel intensity (g) and mean pixel intensity (h) of immunodetection in nuclei as in (c–e), respectively. Error bars standard error; *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test)