Fig. 2.

Cardiac progenitor and cardiomyocyte formation require pSmad1/5, pSmad2/3 and WNT signaling. (A) Barplots showing the expression of Wnt3, Nodal and Eomes in day 1 (left) and day 2 (right) ME WT cells (control) or in cells treated with 1 μM LDN193189 (LN), 10 μM SB431542 (SB), a combination of LN and SB, or in Smad4 KO cells. For all qRT-PCR experiments total RNA was used, expression levels were calculated relative to the corresponding WT control (n=3). (B) Barplot displaying the expression of Nodal, Eomes or T in day 3 ME WT cells (control) or in cells treated with 10 μM XAV939 (XAV) or a combination of 1 μM LN, 10 μM SB and 5 μM CHIR99021 (LN/SB/CHIR) (n=3). (C) Western blot analysis of Eomes or T protein expressed in cells treated as in B. Asterisk indicates a background band. (D) Barplots of Eomes or T expression in day 3 ME WT cells (control) or in cells treated with 1 μM LN alone or with 5 μM CHIR (LN or LN/CHIR), with 10 μM SB alone or with 5 μM CHIR (SB or SB/CHIR). (E) Western blot analysis of Eomes or T protein expression in cells treated as in D. (F) Barplots showing the expression of Mesp1 (day 4) or Actc1 (day 7) in ME WT cells (control) or in cells treated with 1 μM LN alone or in combination with 5 μM CHIR (LN or LN/CHIR), with 10 μM SB alone or with 5 μM CHIR (SB or SB/CHIR), with a combination of 1 μM LN, 10 μM SB and 5 μM CHIR (LN/SB/CHIR), or with 10 μM XAV. (G) Barplot showing the fractions of contracting aggregates (% of total aggregates) at day 8 of differentiation. Cells were treated as in F and grown in 24-well plates. All aggregates in six wells were counted per treatment condition (n=6). See also Fig. S2. Data are mean+s.e.m. *P<5e-2, **P<1e-2 (paired two-tailed Student's t-test). n.s., not significant.