| In Vitro |
Embryos treated with SJ000291942 display the most severe ventralization and SJ000291942 is also the most potent. SJ000291942 also causes more mortality, and at lower doses than controls and the other two compounds. This demonstrates our compounds cause ventralization of embryos consistent with increased BMP signaling activity. SJ000291942 causes an increase in bmp2b and szl expression. Zebrafish assays suggest that SJ000291942 activates the canonical BMP signaling pathway. To extend these observations, immunoblotting of protein lysates from C33A-2D2 cells stimulated with SJ000291942 at different times is performed. SJ000291942 activates phosphorylation of SMAD1/5/8 in serum-free medium. Like in zebrafish embryos, SJ000291942 is most active. SJ000291942 induces p-SMAD1/5/8 maximally at 1hr of treatment. Immunoblotting analysis of lysates from C33A-2D2 treated with SJ000291942 reveals clear induction of the phosphorylated Extracellular Signal-regulated protein Kinase, ERK1/2 (P-ERK1/2) by SJ000291942. The highest dose (100 and 300ng) BMP4 treatments generate a gene expression signature most similar to osteoblast expression. Low dose (10ng) BMP4 treatment aligns closely with 25μM compound 3 treatment and with 25μM SJ000291942[1].
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