Figure 1.

Effects of ATR‐101 versus PD129337 on cholesterol accumulation, cholesterol esterification, ATP levels and caspase 3/7 activities in ACC‐derived cells. (A) Time‐dependence of the effect of ATR‐101 on cholesterol levels in H295R cells. The cells were cultured with DMSO vehicle or with 60 μM ATR‐101 for the times indicated above the images. The images show filipin III binding to cholesterol in fields containing 5500–7800 cells. The scale bars denote 100 μm. The fluorescence intensities of the cell populations and the statistical significance of the differences are shown in Supporting Information Fig. S1A. (B) Time‐dependence of the effects of ATR‐101 on the ATP levels and on the caspase 3/7 activities in H295R cells. The cells were cultured with DMSO vehicle or with 100 μM ATR‐101 for the indicated times. The ATP levels (left graphs) and the caspase 3/7 activities (right graphs) were measured in cells that were grown in parallel. The graphs show the means ± 2SD of five samples from three experiments. *P < 0.05, significantly different from DMSO control; two‐way ANOVA with Sidak's post hoc tests. (C) Effects of ATR‐101 versus PD132997 on the cholesterol levels in H295R and BD140C cells. H295R (upper images) and BD140C (lower images) cells were cultured with DMSO vehicle, ATR‐101 or PD129337 at the indicated concentrations for 4 h. The images show filipin III binding to cholesterol and are representative of images collected in two separate experiments for each cell line. The scale bars denote 10 μm. The full fields from which the images were cropped are shown in Supporting Information Fig. S1D. (D) Effects of different concentrations of ATR‐101 versus PD132997 on the ATP levels and caspase activities in H295R and BD140C cells. H295R (upper panels) and BD140C (lower panels) were cultured with the indicated concentrations of ATR‐101 or PD129337 for 4 h. The ATP levels (left graphs) and the caspase 3/7 activities (right graphs) were measured in cells that were grown in parallel. The graphs show the means ± 2SD of eight samples from four experiments and five samples from three experiments in H295R and BD140C cells respectively. *P < 0.05, significantly different from PD129337; two‐way ANOVA with Sidak's post hoc tests. The ATP levels and the caspase activities of H295R and BD140C cells that were cultured with ATR‐101 or PD132997 for 24 h are shown in Supporting Information Fig. S1B. (E) Effects of ATR‐101 or PD129337 on cholesterol esterification in H295R and BD140C cells. H295R (upper images) and BD140C (lower images) cells were cultured with DMSO vehicle or with the indicated concentrations of ATR‐101 or PD129337 for 2 h, followed by an additional 2 h after the addition of 1 μg·mL⁻¹ NBD‐cholesterol. The images show NBD‐cholesterol ester (green) and Hoechst (blue) fluorescence and are representative of images collected in five separate experiments for each cell line. The scale bars denote 30 μm. The effects of different concentrations of ATR‐101 and of PD129337 on cholesterol esterification are shown in Supporting Information Fig. S1C. The full fields from which the images were cropped are shown in Supporting Information Fig. S1E.