Figure 6. FKHR-PAX3 promoted low-density cell proliferation and blocked terminal myogenic differentiation.

(A) Comparison of cell proliferation in NIH3T3, RD, RH4, and RH30 cultures with or without FKHR-PAX3 (FP3) expression. Cells were seeded in triplicate at 1 x 10⁴ cells/well into a 24 well-plate. Proliferation was measured daily by counting the number of live cells (trypan blue-negative) over five days beginning a day after the initial seeding. Cell death was minimal in all experiments. (B) The effect of FKHR-PAX3 expression on low-density RD and RH30 growth. Cells were seeded in triplicate at 2 x 10³ cells/well into a 24 well-plate. Cell growth was quantified daily using the WST-1 cell proliferation kit. (C) Top panel: Immunodetection of MyoG and MHC expression in proliferating (GM) and differentiated (five days, DM) C2C12 cells with or without FKHR-PAX3. Bottom panel: light (left panel, 100X magnification) and fluorescent (middle and right panels, (250X magnification) microscopic images of day-5 differentiated cells stained with MF20 antibody against MHC (middle) or with DAPI (right). (D) The effect of PAX3-FKHR knockdown on MyoG and MHC expression in control and FKHR-PAX3 expressing RH30 cells. Cell extracts were analyzed by western blot as in Figure 2D. (C-D) Alpha-tubulin was used to normalize sample loading. Representative data from FKHR-PAX3 isoform c is shown.