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. 2022 Oct 5;14(19):4858. doi: 10.3390/cancers14194858

Figure 4.

Figure 4

GlcSph induces necrosis and produces multinucleated cells in A375 human melanoma cells. (A) A375 cells were treated for 24 h with either different concentrations of GlcSph (1, 2.5, and 5 µM) or with ethanol (veh), stained with Annexin V-FITC and 7-AAD, and analyzed by flow cytometry; 20,000 events were recorded and sorted as follows: live cells (Annexin V-negative/7-AAD-negative), dead cells (Annexin V-positive/7-AAD-positive), early apoptosis (Annexin V-positive/7-AAD-negative), and necrosis (Annexin V-negative/7-AAD-positive). Histograms represent the cell frequency of GlcSph-treated cells compared to vehicle-treated cells. (B–E) A375 cells were treated for 48 h with ethanol (B,D) or GlcSph 5 µM (C and E), fixed and stained with phalloidin (for F-actin, green), and nuclei were counterstained with DAPI (for DNA, blue). Then, they were imaged by confocal microscopy (Zeiss) at 63× (B,C), with an additional upper zoom (×2) of the selected regions (white square) (D,E). Scale bar = 20 µm. (F–H) A375 cells were incubated either with different concentrations of GlcSph (1, 2.5, or 5 µM) or with ethanol (veh) for 24, 48, and 72 h. Then, cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Figure (F) is a representative plot of cell cycle analysis of GlcSph- and vehicle-treated A375 cells for 48 h. To identify the peaks corresponding to abnormal ploidies, A375 cells were treated with blebbistatin (10 µM), a myosin II inhibitor (see arrows). The proportion of cells with 8N (G) and 16N (H) in GlcSph-treated cells compared to controls was quantified. For experiments in A, G, and H, data are expressed as means +/− SEM of three independent experiments. Statistical significance was determined by a two-way mixed ANOVA. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 compared to vehicle-treated cells.