Figure 3
Formation of cytoplasmic lipid bodies and production of β-casein in MDA-MB-231 cells exposed to D609. (a) Confocal laser scanning microscopy analyses (three-dimensional reconstruction images) of MDA-MB-231 cells cultured in the absence (CTR) or presence of D609 (50 μg/mL) for the indicated times and stained with Bodipy 493/503 (green). Morphological changes of actin cytoskeleton were monitored by phalloidin-633 staining (red); nuclei are reported in blue (DAPI). Scale bar, 20 μm. (b) Histogram showing the fold increase in Bodipy 493/503 fluorescence intensity measured by flow cytometry in D609-treated MDA-MB-231 cells in comparison with CTR (mean ± standard deviation, n = 3). (c) Changes in β-casein expression detected by Western blot in MDA-MB-231 cells after incubation with D609 for the indicated times (actin as loading control). (d) Representative proton nuclear magnetic resonance (1H NMR) spectrum of intact MDA-MB-231 cells incubated for 48 hours in the absence (black) or presence (green) of D609, showing increases in -(CH2)n- (1.30 ppm) and = CH-CH = (5.34 ppm) signals of mobile lipids in D609-treated cells (details in inserts). Asterisk indicates unidentified signal of commercial phosphate-buffered saline buffer. (e) Thin-layer chromatography analysis of total lipid extracts of D609-treated MDA-MB-231 cells in comparison with CTR. Fold increases were as follows: triacylglycerols (TAG), 1.8 ± 0.1 at 48 hours and 1.8 ± 0.8 at 72 hours (n = 3); cholesteryl esters (CE), 1.4 ± 0.1 at 48 hours and 1.7 ± 0.5 at 72 hours; cholesterol (CHOL), 1.0 ± 0.1 at 48 hours and 1.1 ± 0.1 at 72 hours. Phospholipids (PL) were not significantly altered. CTR, control; DAPI, 4',6-diamidino-2-phenylindole; ppm, parts per million.