Activin-mediated TGF-β, PDGF, and FGF signaling are important for MSC differentiation. Marrow-derived MSCs were cultured in the presence of inhibitors of these pathways. (A) Western blot of MSCs cultured in the presence of SU5402 probed with phospho-FGFR-Y766 antibody. Untreated MSCs as well as MSCs cultured in the presence of tyrphostin AG 370 and SB431542 show the presence of the phosphoyrlated FGFR, whereas cells cultured in the presence of SU5402 do not show phospho-FGFR. SU5402 is reported to be a weak inhibitor of tyrosine phosphorylation of the PDGF receptor and does not inhibit phosphorylation of the TGF-β receptor. The Western blot in this figure confirms this. (B) MSCs were cultured in differentiation medium for 14 days and stained with Oil Red O, Alcian Blue, and Von Kossa for staining adipocytes, chondrocytes, and osteocytes, respectively. Inhibition of any of these 3 pathways leads to an altered differentiation potential of MSCs, proving that these pathways are essential for MSC differentiation. Oil Red O and Von Kossa micrographs aquired with a Zeiss Axiovert 40 C microscope (Carl Zeiss, Singapore) fitted with a 20×/0.3 NA objective. Alcian Blue micrographs were acquired with a 10×/0.25 NA objective on the same microscope. All images were captured on a 7.1 megapixel Canon Powershot A620 camera (Canon, Singapore) and the contrast was adjusted using the auto-contrast feature in Picasa (Google, Mountain View, CA).