Figure 2. Bexarotene inhibits cardiac differentiation of P19 pluripotent stem cells.

(A) P19 cells were differentiated with DMSO in the absence or presence of increasing concentrations of bexarotene (BEX, 10 nM, 100 nM, 1 μM) or RA (10 nM) during EB formation. The cells were then maintained on coverslips without any treatments for an additional 5 days and costained with specific antibodies for MyoD (red), myosin heavy chain (MyHC, green) and Hoechst for the nuclei (blue). The cells were also harvested at different time points for RT-PCR and Western analyses. (B) Shown are the representative images of microscopy. (C) Quantification of differentiation is presented as the fractions of cardiac myocytes (car, dark grey) and skeletal myocytes (sk, light grey) in relation to the total cell population (n = 5). (D) Gene expression of myogenin and GATA4 and protein on day 4 and day 9 of differentiation was determined by Western analysis in parallel with the use of the same batch of cell extracts. The blots were then stripped and reprobed for β-tubulin as loading controls. Shown are the cropped blot images representing indicated protein. (E) and (F) Real-time RT-PCR was used to determine the transcripts levels of Pax3 and Meox1 on day 4 of differentiation in the same batch of cDNA. Quantification is presented as fold changes in reference to the untreated EBs (n = 3).