Augmented TGF-β1 and JNK signaling in RUNX-1^+/− iHPCs and iMks. (A-B) ELISA of TGF-β1 levels in iMk culture conditioned medium. Cell culture supernatants were collected from L1-C and L1 day 13 iMk liquid cultures and used to measure levels of active TGF-β1. Mean ±1 SEM of TGF-β1 without normalization for differences in the number of iMks (A) and with normalization for iMks (B) (n = 5 experiments per study arm). (C) The effect of TGF-β1 exposure on Mk yield from L1-C and L1 iHPCs. Cultures were exposed to rhTGF-β1 for 5 days at the indicated doses, and Mk yield was quantified per input iHPC, as in Figure 1C. Results are shown as a percentage of untreated iMks. (D) The effect of 20 µg/mL anti-α-TGF-β–blocking antibodies (Abs) on suppression of Mk yield resulting from TGF-β1 exposure. Mean ±1 SEM (n = 6 experiments per study arm; P values calculated by 2-way analysis of variance. (E) Day 13 iMk proteins with antibodies directed against TGFβR-1, p21, p57, and pSMAD2/3 with GAPDH as a loading control. (F) Day 7 iHPC immunostained with antibodies against phosphorylated JNK (pJNK). Mean ±1 SEM (n = 4 experiments per study arm). (G) Elevated JNK phosphorylation in day 13 L1 iMks. The iMks were solubilized and immunoblotted with antibodies against pJNK, total JNK, and vinculin (as a loading control). (H) Experiments in panel G were scanned and optical density was quantified. Mean ±1 SEM (n = 6 experiments per study arm). All P values by Student t test, unless stated otherwise.