Fig. S2

Fibronectin promotes End2-mediated induction of mesoderm and precardiac mesoderm in vitro and in vivo. (A) Knockdown efficiency of the indicated siRNAs assayed by qPCR. (B) Percentage of Bry-GFP+ cells in day-4 EBs cultured with End2 cells with control (IgG) or anti-Fn1 antibody. (C) EBs with increasing ratio of End2 cells stained with DAPI and Fn1 antibody. (D) Adherent culture of mESBry-GFP EBs plated with End2 cells and stained for Fn1 (red) and GFP (green). (E) End2 cells 48 hours after transfection with scrambled siRNA or siRNA against Fn1, stained for Fn1 (green) and β-catenin (red). (F) End2 cells 48 hours after transfection with scrambled siRNA or siRNA against Fn1, stained for pan-cadherin (green) and aPKC (red). (G) EBs containing End2 cells transfected with scrambled siRNA or siRNA against Fn1, stained for pan-cadherin (green) and aPKC (red). (H) FACS analysis of cell proliferation as assayed by Ki-67 or phosphohistone H3 (PH3) staining among Bry-GFP+ cells in EBs aggregated with control or fibronectin-deficient End2 cells. (I) FACS analysis of cell death as assayed by propidium iodide (PI)+ and annexin V staining among Bry-GFP+ populations in EBs aggregated with control or fibronectin-deficient End2 cells. (J) Expression of the pluripotent genes Oct4 and Nanog in day-2 EBs with control or Fn1-deficient End2 cells. (K) Scatter plot of ECM mRNA array from EBs co-cultured with control or Fn1 knockdown End2 cells. (L) Whole-mount in situ hybridization of ntl in control or Fn1/Fn3 knockdown (MO-Fn1/3) zebrafish embryos at 90% epiboly (9 hpf). *P<0.05, error bars indicate s.e.m.