Y-27632 treatment of P19 cells inhibits the differentiation of primitive endoderm.

<p>Monolayer grown P19 cells induced to differentiate with RA were collected for mRNA quantification and immunofluorescence. Total mRNA from all treatment groups was probed with a GATA-6 primer/probe, which is a marker of primitive endoderm. The RA treatment group displayed an increase of GATA-6, but decreased when RA was supplemented with Y-27632, with moderate recovery when cells where induced to differentiate after Y-27632 pre-treatment and removal of the inhibitor (A). Immunofluorescence of GATA-6 in undifferentiated cells (B) and in RA treated (C), RA+Y-27632 treated (D) or RA+Y-27632 pre-treated cells. Total mRNA converted to cDNA and probed with Troma-1, showed a dramatic decrease in expression when Y-27632 was present either during or before differentiation (F). Immunofluorescence of Troma-1 in undifferentiated (B) RA treated (C), RA+Y-27632 treated (D) and RA+Y-27632 pre-treated cells. FACS analysis demonstrates a significant decrease in GATA-6 and Troma-1 positive cells with Y-27632 present (K). Scale bars equals 50 µm, green staining represents the primary antibody, and blue staining is the nuclear dye TOTO-3. * Significance accepted at p<0.05.