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Fig. 3 | BMC Cancer

Fig. 3

From: Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage

Fig. 3

Cell death induced by HMA. Involvement of RIPK3. a PI (Propidium Iodide) permeability of untreated and HMA-treated (10-40 μM, 24 h) HCT-116 cells. Cytofluorimetric analysis allowed the quantification of PI+/dead cells detected on 10,000 events (mean ± s.d. calculated on three independent experiments). **P < 0.01, ***P < 0.001. b Cytofluorimetric analysis of untreated and HCT-116 cells treated with HMA alone (30 μM and 40 μM) or together with 20 μM pancaspase inhibitor zVAD.fmk or necroptosis inhibitor Necrostatin-1 (NEC, 50 μM); parallel samples were incubated with zVAD.fmk or NEC alone. Cells were stained with PI; values are expressed as percentage of PI positive events on 10,000 events recorded calculated on three independent experiments. *P < 0.05, ***P < 0.001, n.s. not significant. c Western blot analysis of necroptosis proteins RIPK1, RIPK3 and MLKL in HT-29 cells untreated and treated with 30 μM HMA for 24 h. α-Tubulin: loading control. d Effect of silencing of RIPK3 by siRNA on HT-29 cell survival after a 30-μM HMA treatment (for 24 h). Cell death was monitored by flow cytometry after Annexin V/PI staining; four fractions can be distinguished: PI+/A+: late apoptotic/necrotic cells; A+: early apoptotic cells; PI+: necrotic cells; unstained: alive cells. e Western blot analysis of RIPK3 in HT-29 cells after transfection with siRNAs; γ-tubulin was used as loading control; red rectangle refers to the siRNA used in d). A representative experiment out of three is shown in panels (c) and (e)

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