Fig. 4

PMA suppresses both adipocyte differentiation and the proliferation of BMSCs under both adipogenesis and high-Ca²⁺ conditions. a BMSCs were cultured in standard medium. After 7 days, the cells were cultured for 1 day in adipocyte differentiation medium with addition of 9 mM CaCl₂ or 100 nM ionomycin and/or 100 nM PMA. Total RNA was isolated, and the mRNA levels of C/EBPα and PPARγ were determined using real-time quantitative RT-PCR (n = 4). b–d BMSCs were cultured in adipocyte differentiation medium for 14 days with addition of 9 mM CaCl₂ or 100 nM ionomycin and/or 100 nM PMA. (b-top) The cell numbers were evaluated using a modification of the MTT assay (n = 9). (b-bottom) The cell numbers were counted using a hematocytometer (n = 4). c Cell viability was measured by propidium iodide (PI) uptake and flow cytometry. A representative histogram of cells is shown in the top panel, and the cell viability is shown in the bottom panel (n = 4). d Typical photomicrographs of Oil Red O staining of cells are shown in the upper panels. Bars indicate 100 μm. The levels of total adipocyte accumulation were measured based on Oil Red O extraction and shown in the bottom panel (n = 9). a–d^*P < 0.05, ^**P < 0.01 vs. control without PMA, ^†P < 0.05, ^††P < 0.01 vs. + 9 mM CaCl₂ without PMA, ^§P < 0.05, ^§§P < 0.01 vs. + 100 nM ionomycin without PMA