Pannexin-1 controls the Uptake of TO-PRO-3 during Necroptosis.Figure 2A HT-29 cells were transfected with two individual siRNA for PANX1, or scramble non-specific (NS) siRNA for 72 hours. Cells were pre-treated with 10 μM QVD-OPh (Q) together with 5 μM Birinapant (S), prior stimulation with 10 ng.mL-1 of TNFα (T), as indicated. Western blotting for hallmarks of necroptosis, as indicated. The arrowhead indicates MLKL cleaved fragment. Molecular weight markers (Mr) are shown. B Cells were exposed to TQS for 5 hours. Necrostatin-1 (Nec-1s, 20 μM) was also used. MLKL oligomers (MLKLn) were resolved by non-reducing SDS-PAGE after cross-linking. C The cell viability was assessed by CellTiter-Glo after 24 hours of treatment (means ± SEM, n=4 biological replicates). Flow cytometric analysis of cells treated with TQS for 4 hours and stained with Annexin V (D) Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA). Flow cytometric analysis of cells treated with TQS for 4 hours and stained with TO-PRO-3 (E and F). Data are means ± SEM of three independent experiments. *P<0.1, ****P<0.0001 (ANOVA). HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death, and analyzed by Western blotting (G). HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death Cell survival was assessed by CellTiter-Glo after 24 hours of treatment (H). Histogram shows means ± SEM, n=4 biological replicates, ****P<0.0001, ANOVA. HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated as in (A) with the addition of 1 μM cycloheximide (C) to enhance cell death Annexin V staining was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA. HT-29 cells were infected with lentiviruses containing two individual sgRNA to deplete endogenous PANX1. A control nonspecific sequence (NC) was also used. Cells were treated with the addition of 1 μM cycloheximide (C) to enhance cell death TO-PRO-3 uptake was analyzed by flow cytometry after 4 hours of stimulation Shown are means ± SEM, n=3 biological replicates, *P<0.1, ****P<0.0001, ANOVA. PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. Cell lysates were analyzed by Western blotting as indicated (K). PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. TO-PRO-3 uptake was analyzed by flow cytometry Shown are means ± SEM, n=3 biological replicates, ****P<0.0001 ANOVA. PANX1 expression was restored in PANX1 knockout cells after an infection with a lentivirus containing a PANX1 cDNA and expressing GFP. TO-PRO-3 uptake was analyzed by flow cytometry Shown are means ± SEM, n=3 biological replicates, ****P<0.0001 ANOVA. NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated Cell survival was evaluated by CellTiter-Glo after 24 hours (N). Data are means ± SEM, n=4 biological replicates, ***P<0.001, ****P<0.0001, ANOVA. NS- and MLKL-silenced cells were pretreated with 100 μM carbenoxolone (CBX), 2 mM Probenecid (Probe) or 30 μM Trovafloxacin (Trova) and stimulated TO-PRO-3 uptake was analyzed by flow cytometry (O). Data are means ± SEM, n=3 biological replicates, ***P<0.001, ****P<0.0001, ANOVA. P Cells were analyzed by Western blotting. The presented data are representative of at least three independent experiments.