(A) Schematic timeline of MSN differentiation. (B) qPCR data from RNA extracted at 19DIV after treatment with Activin (25 ng/ml) alone or plus SB431542 (10 µM) as shown in panel (A). Data show mean RQ ± SEM relative to control (n = 6, 6 and 2, respectively). (C) qPCR data from RNA extracted 1 or 6 days after treatment that began at 10DIV. Cells were untreated (control) or treated with Activin (25 ng/ml), or Activin plus SB525334 (1 µM), LY2109761 (5 µM) or LDN193189 (100 nM). Data show mean RQ ± SEM relative to the 10DIV+1 control (n = 2 per group). (D) A repeat of the experiment in panel (C) with just control, Activin and Activin plus LDN conditions to confirm the LDN effects on CTIP2 and GSH2 up-regulation. Data show mean RQ ± SEM relative to the 10DIV+1 control (n = 3 per group; *P<0.05, **P<0.01, ***P<0.001; two-way ANOVA with post-hoc Bonferroni).