Cholesterol inhibits cell death induced by GalSph and GlcSph in MCF7 cells.

(A-C) Death of MCF7 cells treated for 48 hours with indicated concentrations of GalSph (A), GlcSph (B), or ebastine (C) was determined as in Fig 1A. When indicated, 60 μM cholesterol was added 24 hours before the addition of lysosphingolipids or CADs. (D) Percentage of galectin-3 puncta positive MCF7 galectin-3-eGFP cells treated as indicated with GlcSph and visualized as in (E). When indicated, 60 μM cholesterol was added 24 hours before the addition of lysosphingolipids (n = 2). (E) Representative images of MCF7 galectin-3-eGFP cells treated with indicated concentrations of GlcSph, for 17.5 hours were acquired using the ImageXpress (FITC channel). When indicated, 60 μM cholesterol was added 24 hours before the addition of GlcSph. Scale bars, 10 μm (F-G) Quantities of HexSph (GalSph and GlcSph) in lysates of MCF7 cells treated with 45 μM GalSph (F) or 32 μM GlcSph (G). When indicated, 60 μM cholesterol was added 24 hours before the addition of lysosphingolipids. Error bars, SD of three independent experiments. *, P P P < 0.0001 as analyzed by two-way ANOVA followed by Sidak’s multiple comparisons tests.