Extended Data Fig. 2: Mesoderm derivation.
a, Immunoblots showing expression of SMAD2, SMAD3, pSMAD3, and SMAD4 in the mesoderm cells. Quantifications represent mean ± SEM; pSMAD3 was increased in SMAD2−/− (P = 0.0396) and SMAD2mh1/mh1 (P = 0.0076) mesoderm cells. SMAD3 was increased in SMAD2−/− (P = 0.0874) and SMAD2mh1/mh1 (P = 0.0164) mesoderm cells. n = 3 independent experiments. b, Schematic representation of the derivation of posterior mesoderm cells. c, Representative phase contrast images of WT, SMAD2−/−, and SMAD2mh1/mh1 iPS cell-derived posterior mesoderm. Scale bar, 275 µm. d, Immunoblots showing expression of mesoderm lineage-specific markers in the differentiated mesoderm derived with or without Activin A pulses. n = 2 independent experiments. e, RNA expression of posterior mesoderm-specific genes. Data are mean values ± SEM; n = 3 independent experiments. f, Quantification of cell-secreted Activin A on Days 1 and 2 during posterior mesoderm induction. Data are mean values ± SEM; n = 2 independent experiments. We performed a P < 0.05 one-way ANOVA with Dunnett’s multiple comparison test (a, e). Color symbols shown in the histograms of Figs. a and e represent a biological replicate. Same colors in Fig. e indicate 3 independent wells of mesoderm cells from each biological replicate. Representative images from 3 independent experiments are shown in Fig. c. Images of all the stain-free gels and stain-free blots can be found in Supplementary Fig. 6a. Source data are available for this figure.
