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Necroptosis characterization during infection with oncolytic rVSV or rVSV-NDV in A549 and H1437 cells A549 and H1437 cells were infected with either rVSV or rVSV-NDV at an MOI of 1. Lysates were collected at 8, 16, and 24 hpi and analyzed by western blot for the expression and phosphorylation of necrosome components RIPK1, RIPK3, or MLKL. Representative western blots of OV-infected (A) A549 and (C) H1437 were cropped at the solid vertical lines, and arrowheads on the right indicate the quantified protein bands. For simplicity, only one representative GAPDH blot is shown. Corresponding quantification of RIPK1, RIPK3, or MLKL phosphorylation was derived from the protein expression of unphosphorylated and phosphorylated protein relative to the respective GAPDH expression and is visualized in (B and D) as the phosphorylation fold-change relative to untreated cells. Representative blots of n = 3 independent biological replicates are plotted and mean (± SD) values of n = 3 independent biological replicates are shown.

Journal: Molecular Therapy Oncology

Article Title: Syncytial death mediated by oncolytic rVSV-NDV dynamically activates immunogenic apoptosis and necroptosis in human lung cancer cells

doi: 10.1016/j.omton.2025.201027

Figure Lengend Snippet: Necroptosis characterization during infection with oncolytic rVSV or rVSV-NDV in A549 and H1437 cells A549 and H1437 cells were infected with either rVSV or rVSV-NDV at an MOI of 1. Lysates were collected at 8, 16, and 24 hpi and analyzed by western blot for the expression and phosphorylation of necrosome components RIPK1, RIPK3, or MLKL. Representative western blots of OV-infected (A) A549 and (C) H1437 were cropped at the solid vertical lines, and arrowheads on the right indicate the quantified protein bands. For simplicity, only one representative GAPDH blot is shown. Corresponding quantification of RIPK1, RIPK3, or MLKL phosphorylation was derived from the protein expression of unphosphorylated and phosphorylated protein relative to the respective GAPDH expression and is visualized in (B and D) as the phosphorylation fold-change relative to untreated cells. Representative blots of n = 3 independent biological replicates are plotted and mean (± SD) values of n = 3 independent biological replicates are shown.

Article Snippet: Membranes were blocked using 5% skim milk powder (Carl Roth) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for at least 1 h at room temperature and incubated with the following primary antibodies in 2.5% BSA in TBST at 4°C overnight: mouse anti- caspase-3 (31A1067) (#56053, Santa Cruz Biotechnology), mouse anti- caspase-8 (1C12) (#9746, Cell Signaling Technology [CST]), rabbit anti-caspase-9 (#9502, CST), rabbit anti-cleaved caspase-9 (D315) (#9505, CST), rabbit anti-PARP antibody (#9542, CST), rabbit anti-human phospho RipK1 (S166) (D1L3S) (#65746, CST), mouse anti-RIP (clone 38/RIP) (#610459, BD Biosciences), rabbit anti-human RIPK3 antibody (#226297, Abcam), rabbit anti-RIPK3 (phospho S227) (EPR 9627) (#209384, Abcam), rabbit anti-MLKL (phospho S358) antibody [EPR9514] (#187091, Abcam), and rabbit anti-MLKL antibody [ EPR17514 ] (#184718, Abcam).

Techniques: Infection, Western Blot, Expressing, Phospho-proteomics, Derivative Assay

Impact of genetic deletion of upstream cell death mediators caspase-8, RIPK1, or RIPK3 on rVSV- or rVSV-NDV-mediated oncolysis in A549 and H1437 cells Gene knockouts for caspase-8, RIPK1, or RIPK3 and two NTCs were generated using CRISPR-Cas9 technology. Validated single-cell knockout clones of (A and B) A549 and (C and D) H1437 cells were infected with GFP-expressing rVSV or rVSV-NDV at an MOI of 0.01, and changes in cell viability were measured by CellTiter-Glo assay in a time course experiment. (A and C) Viability data were normalized to uninfected cells and expressed as log2-fold changes to the average of NTC1 and NTC2. Mean values (± SD) of n = 3 (A549) or n = 4 (H1437) independent biological replicates are plotted. Representative fluorescent and brightfield microscopy images ( n = 3 independent biological replicates) of OV-infected (B) A549 or (D) H1437 knockout cells are shown. Microscopy images have been adjusted to similar contrast and brightness.

Journal: Molecular Therapy Oncology

Article Title: Syncytial death mediated by oncolytic rVSV-NDV dynamically activates immunogenic apoptosis and necroptosis in human lung cancer cells

doi: 10.1016/j.omton.2025.201027

Figure Lengend Snippet: Impact of genetic deletion of upstream cell death mediators caspase-8, RIPK1, or RIPK3 on rVSV- or rVSV-NDV-mediated oncolysis in A549 and H1437 cells Gene knockouts for caspase-8, RIPK1, or RIPK3 and two NTCs were generated using CRISPR-Cas9 technology. Validated single-cell knockout clones of (A and B) A549 and (C and D) H1437 cells were infected with GFP-expressing rVSV or rVSV-NDV at an MOI of 0.01, and changes in cell viability were measured by CellTiter-Glo assay in a time course experiment. (A and C) Viability data were normalized to uninfected cells and expressed as log2-fold changes to the average of NTC1 and NTC2. Mean values (± SD) of n = 3 (A549) or n = 4 (H1437) independent biological replicates are plotted. Representative fluorescent and brightfield microscopy images ( n = 3 independent biological replicates) of OV-infected (B) A549 or (D) H1437 knockout cells are shown. Microscopy images have been adjusted to similar contrast and brightness.

Article Snippet: Membranes were blocked using 5% skim milk powder (Carl Roth) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for at least 1 h at room temperature and incubated with the following primary antibodies in 2.5% BSA in TBST at 4°C overnight: mouse anti- caspase-3 (31A1067) (#56053, Santa Cruz Biotechnology), mouse anti- caspase-8 (1C12) (#9746, Cell Signaling Technology [CST]), rabbit anti-caspase-9 (#9502, CST), rabbit anti-cleaved caspase-9 (D315) (#9505, CST), rabbit anti-PARP antibody (#9542, CST), rabbit anti-human phospho RipK1 (S166) (D1L3S) (#65746, CST), mouse anti-RIP (clone 38/RIP) (#610459, BD Biosciences), rabbit anti-human RIPK3 antibody (#226297, Abcam), rabbit anti-RIPK3 (phospho S227) (EPR 9627) (#209384, Abcam), rabbit anti-MLKL (phospho S358) antibody [EPR9514] (#187091, Abcam), and rabbit anti-MLKL antibody [ EPR17514 ] (#184718, Abcam).

Techniques: Generated, CRISPR, Knock-Out, Clone Assay, Infection, Expressing, Glo Assay, Microscopy