Fig. 1. Effects of high-dose GluOC on the morphology and number of 3T3-L1 adipocytes.

a Representative phase-contrast microscopic images of 3T3-L1 adipocytes or preadipocytes cultured with GluOC (5 or 40 ng/ml) or with 1 μM staurosporine (STS) for 48 or 96 h. Scale bars, 200 µm. b Cell counts determined from images as in a. Data are expressed as a percentage of the initial value and are means + SEM from three independent experiment. **p < 0.01 versus the corresponding value for vehicle-treated (control) cells (one-way ANOVA followed by the Tukey–Kramer HSD test). c Immunoblot analysis of PCNA and β-actin (loading control) in 3T3-L1 adipocytes cultured with the indicated concentrations of GluOC for 24 or 48 h. The blot is representative of three independent experiments. d Fluorescence and phase-contrast microscopy of 3T3-L1 adipocytes cultured with the indicated concentrations of GluOC or with 1 μM staurosporine for 48 h. Nuclei were stained with Hoechst 33342 (green fluorescence). Arrows in overlay images indicate adipocytes showing smaller lipid droplets and collapse of the plasma membrane, arrowheads indicate cells showing nuclear swelling and loss of the plasma membrane, and the asterisk indicates adipocytes with a morphology similar to those of vehicle-treated cells. The right panel shows an enlarged image of the overlay panel for GluOC (40 ng/ml). Scale bars, 50 µm. Images are representative of three independent experiments. e Time-lapse phase-contrast microscopy (Keyence BZ-X700 microscope) of 3T3-L1 adipocytes cultured with the indicated concentrations of GluOC for up to 48 h. Images were captured at 1-h intervals. Arrowheads (blue, green, or red) indicate representative single-cell dynamics. The right panels show enlarged areas of the corresponding left panels. Scale bars, 30 µm