Figure 2

PC-PLC and SMS activity inhibition and cell growth arrest in MDA-MB-231 cells exposed to D609 (50 μg/mL). (a) Absolute PC-PLC activity (mean ± SD, n = 4) measured in total lysates of MDA-MB-231 cells harvested after exposure to D609 (gray columns) in comparison with untreated cells (CTR, black columns). Student t test, *P ≤ 0.04, **P ≤ 0.02, ***P = 0.0002; ns, not significant. (b) Cell counts (mean ± SD, n = 3) of MDA-MB-231 cells incubated in the absence (black square, viable cells; white square, dead cells) or presence (black circle, viable cells; white circle, dead cells) of D609. Cells were seeded (S) 48 hours before treatment, which started at t = 0 (arrow). (c) Percentage of Annexin V-positive cells in D609-treated and the respective CTR cells. (d) Relative SMS activity detected in MDA-MB-231 cells incubated in the absence (CTR) or presence of D609 for the indicated times. Lipid extracts were added with BTR-ceramide, and the formation of the SMS product, BTR-sphingomyelin (BTR-SM), was detected by thin-layer chromatography and quantified by densitometric analysis (representative example of two independent experiments). (e) Comparison of the percentages of PC-PLC (gray) and SMS (white) inhibition induced in MDA-MB-231 cells by exposure to D609 for 48 or 72 hours. BTR, Bodipy-TR; PCho, phosphocholine; PC-PLC, phosphatidylcholine-specific phospholipase C; SD, standard deviation; SMS, sphingomyelin synthase.