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. 2014 Oct 22;1(10):649–664. doi: 10.18632/oncoscience.89

Figure 5. Induction of Nec-1-inhibitable necrosis in edelfosine-treated U118 cells.

Figure 5

(A) Cell proliferation was measured by MTT assay at the indicated time points, after culturing U118 cells without or with 200 μM Nec-1 (Nec-1) for 2 h, and then incubated in the absence or presence of 10 μM edelfosine (EDLF). Untreated control cells were run in parallel. Data are expressed as means ± SD of at least three independent experiments, each one performed in triplicate. The right plot shows only the measurements for edelfosine- and Nec-1+edelfosine-treated cells for an easier appreciation of changes. ***, P<0.001 EDLF vs. Nec-1+EDLF, Student's t test. (B) Quantification of U118 cells stained with PI after treatment with 10 μM edelfosine (EDLF) for 4 h without and with a pretreatment of 200 μM Nec-1 (Nec-1+EDLF) or 200 μM Nec-1s (Nec-1s+EDLF). Data shown are means ± SD of three independent experiments. ***, P<0.001 Nec-1+EDLF vs. EDLF; ***, P<0.001 Nec-1s+EDLF vs. EDLF, Student's t test. (C) Representative flow cytometry analysis histograms of PI incorporation showing: untretated control cells (Control), 10 μM edelfosine-treated cells (EDLF), and cells treated with Nec-1 (200 μM, 2 h pretreatment) + EDLF (10 μM) (Nec-1+EDLF) for 4 h. (D, upper pannel) Bright-field microscopy of untreated control cells, 10 μM edelfosine treated cells for 4 h (EDLF), and cells preincubated with 200 μM NEC-1 for 2 h and then treated for additional 4 h with 10 μM edelfosine (Nec-1+EDLF). Magnification, 20x. (D, lower pannel) FSC/SSC histograms of the cells treated as in the upper panels, showing cellular size (FSC-H) and granularity (SSC-H). Dead cells show lower FSC than living cells. (E) Cells were preincubated without or with 200 μM Nec-1 (Nec-1) for 2 h, then incubated in the absence or presence of 10 μM edelfosine (EDLF) for 24 h, and analyzed by flow cytometry to evaluate apoptosis. Untreated control cells were run in parallel. Data shown are means ± SD of three independent experiments. ***, P<0.001 EDLF vs. Nec-1+EDLF, Student's t test. (F) Cells were untreated (Control, C), treated with 10 μM edelfosine for 24 h (EDLF), pretreated with 200 μM Nec-1 for 2 h and then incubated with edelfosine for 24 h (Nec-1+EDLF), or treated with 200 μM Nec-1 for 26 h (2 h + 24 h). Cells were then analyzed by immunoblotting using specific antibodies against cleaved caspase-3 and LC3B-I/II. Immunoblotting for β-actin was used as an internal control for equal protein loading in each lane. Data shown in C, D and F are representative of three independent experiments.