Journal: Ecotoxicology and environmental safety

Article Title: Arsenic exposure provoked prostatic PANoptosis by inducing mitochondrial dysfunction in mice and WPMY-1 cells.

doi: 10.1016/j.ecoenv.2025.118139

Figure Lengend Snippet: Fig. 2. NaAsO2 exposure induced ZBP1/RIPK1/RIPK3/p-MLKL-mediated necroptosis in WPMY-1 cells. (A−B) mRNA levels of RIPK1, MLKL, and RIPK3 (A) and immunoblotting of ZBP1, RIPK1, MLKL, p-MLKL, and RIPK3 (B) in WPMY-1 cells after the indicated treatment. (C) Quantification data of B. (D−F) Cell viability (D) and immunoblot of ZBP1, RIPK1, MLKL, p-MLKL, and RIPK3 (E) in WPMY-1 cells pre-incubated with 100 μM Nec-1 for 3 h, followed by 48 h of 4 μM NaAsO2 treatment. (F) Quantification data of E. Results are displayed as the mean ± SE based on three repeated trials. *: P < 0.05, **: P < 0.01.

Article Snippet: A semi-dry transfer system was employed to transfer proteins from a 4–20 % gel to a 0.22 μm BioTraceTM NT NC membrane (66485; Pall, Florida, USA).To block the NC membranes, 5 % nonfat milk was applied for 30 min, followed by a 2 h incubation at room temperature using primary antibodies targeting the translocase of outer mitochondrial membrane 20 (Tomm20; 1:2000; ab56783; Abcam, Cambridge, UK), ZBP1 (1:2000; 13285–1-AP; Proteintech, Wuhan, China), RIPK3 (1:500; 17563–1-AP; Proteintech), MLKL (1:2000; 380559; Zenbio, Chengdu, China), p-MLKL (Ser358) (1:1000; 91689; CST, MA, USA), RIPK1 (1:2000; 17519–1-AP; Proteintech), NLRP3 (1:1000; R381207; Zenbio), caspase-1 (1:1000; P79884–2R; Abmart, Shanghai, China), GSDMD (1:1000; P79887R; Abmart), caspase-3 (1:1500; 19677–1-AP; Proteintech), GSDME (1:2000; ab215191; Abcam), interleukin (IL)-18 (1:2000; R24693; Zenbio), Bax (1:2000; 5023; CST), IL-1β (1:1000; 660092; Zenbio), caspase-8 (1:1000; T55038; Abmart), β-actin (1:10000; Proteintech), and Bcl-xL (1:1000; 2762; CST).

Techniques: Western Blot, Incubation

Journal: Ecotoxicology and environmental safety

Article Title: Arsenic exposure provoked prostatic PANoptosis by inducing mitochondrial dysfunction in mice and WPMY-1 cells.

doi: 10.1016/j.ecoenv.2025.118139

Figure Lengend Snippet: Fig. 4. NaAsO2 exposure triggered mitochondrial damage-activated PANoptosis in WPMY-1 cells. (A) MMP was measured using JC-1 probes (scale bar, 20 μm). (B−C) DCFDA (B) and MitoSOX (C) assays reveal intracellular ROS and mtROS levels in WPMY-1 cells after the indicated treatment. (D−F) Effects of NAC on NaAsO2- triggered apoptosis (D−E) and LDH activity (F). (G) Effects of NaAsO2 and NAC on protein levels of Bcl-xL, Bax, ZBP1, RIPK1, RIPK3, MLKL, p-MLKL, GSDME, caspase-3, GSDME-N, cleaved caspase-3, IL-18, and IL-1β in WPMY-1 cells. (H) Effects of NaAsO2 and Mito-TEMPO on protein levels of ZBP1, RIPK1, RIPK3, MLKL, p- MLKL, GSDME, caspase-3, GSDME-N, cleaved caspase-3, IL-18, and IL-1β in WPMY-1 cells. The corresponding quantification data for G and H are provided in Figs. S3D and S4D, respectively. Results are displayed as the mean ± SE based on three repeated trials. *: P < 0.05, **: P < 0.01.

Article Snippet: A semi-dry transfer system was employed to transfer proteins from a 4–20 % gel to a 0.22 μm BioTraceTM NT NC membrane (66485; Pall, Florida, USA).To block the NC membranes, 5 % nonfat milk was applied for 30 min, followed by a 2 h incubation at room temperature using primary antibodies targeting the translocase of outer mitochondrial membrane 20 (Tomm20; 1:2000; ab56783; Abcam, Cambridge, UK), ZBP1 (1:2000; 13285–1-AP; Proteintech, Wuhan, China), RIPK3 (1:500; 17563–1-AP; Proteintech), MLKL (1:2000; 380559; Zenbio, Chengdu, China), p-MLKL (Ser358) (1:1000; 91689; CST, MA, USA), RIPK1 (1:2000; 17519–1-AP; Proteintech), NLRP3 (1:1000; R381207; Zenbio), caspase-1 (1:1000; P79884–2R; Abmart, Shanghai, China), GSDMD (1:1000; P79887R; Abmart), caspase-3 (1:1500; 19677–1-AP; Proteintech), GSDME (1:2000; ab215191; Abcam), interleukin (IL)-18 (1:2000; R24693; Zenbio), Bax (1:2000; 5023; CST), IL-1β (1:1000; 660092; Zenbio), caspase-8 (1:1000; T55038; Abmart), β-actin (1:10000; Proteintech), and Bcl-xL (1:1000; 2762; CST).

Techniques: Activity Assay

Journal: Ecotoxicology and environmental safety

Article Title: Arsenic exposure provoked prostatic PANoptosis by inducing mitochondrial dysfunction in mice and WPMY-1 cells.

doi: 10.1016/j.ecoenv.2025.118139

Figure Lengend Snippet: Fig. 6. NAC mitigated PANoptosis induced by NaAsO2 in mouse prostate. (A) C57BL/6 mice were orally administered 0, 0.5, and 5 mg/kg NaAsO2, 100 mg/kg NAC, and co-administered NaAsO2 (5 mg/kg) and NAC for 90 days (n = 8). (B−C) Body weight (B) and prostate organ coefficients (C) in mice treated as indicated (n = 8). (D) Arsenic concentration in the prostate of mice following the indicated treatment (n = 3). (E) mRNA levels of ZBP1, RIPK1, MLKL, caspase-3, GSDME, IL-18, and IL- 1β in mouse prostate tissue post-treatments (n = 3). (F) Quantitative analysis of caspase-3, GSDME, ZBP1, RIPK1, RIPK3, MLKL, p-MLKL, cleaved caspase-3, Bax, Bcl- xL, GSDME-N, IL-18, and IL-1β in the prostate of mice subjected to the specified treatments (n = 6–8). The corresponding immunoblot data for F are provided in Fig. S6. Results shown are as the mean ± SE. *: P < 0.05, **: P < 0.01.

Article Snippet: A semi-dry transfer system was employed to transfer proteins from a 4–20 % gel to a 0.22 μm BioTraceTM NT NC membrane (66485; Pall, Florida, USA).To block the NC membranes, 5 % nonfat milk was applied for 30 min, followed by a 2 h incubation at room temperature using primary antibodies targeting the translocase of outer mitochondrial membrane 20 (Tomm20; 1:2000; ab56783; Abcam, Cambridge, UK), ZBP1 (1:2000; 13285–1-AP; Proteintech, Wuhan, China), RIPK3 (1:500; 17563–1-AP; Proteintech), MLKL (1:2000; 380559; Zenbio, Chengdu, China), p-MLKL (Ser358) (1:1000; 91689; CST, MA, USA), RIPK1 (1:2000; 17519–1-AP; Proteintech), NLRP3 (1:1000; R381207; Zenbio), caspase-1 (1:1000; P79884–2R; Abmart, Shanghai, China), GSDMD (1:1000; P79887R; Abmart), caspase-3 (1:1500; 19677–1-AP; Proteintech), GSDME (1:2000; ab215191; Abcam), interleukin (IL)-18 (1:2000; R24693; Zenbio), Bax (1:2000; 5023; CST), IL-1β (1:1000; 660092; Zenbio), caspase-8 (1:1000; T55038; Abmart), β-actin (1:10000; Proteintech), and Bcl-xL (1:1000; 2762; CST).

Techniques: Concentration Assay, Western Blot

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Necrotic-like morphology was observed in PANC1 cells following treatment with CCT137690 (10 μM) for 24 hours. (B) Western blot analysis of indicated proteins in PANC1 and PANC2.03 cells following treatment with CCT137690 (2.5–10 μM) for 24 hours (n=3, *p < 0.05 versus untreated group). (C, D) PANC1 and PANC2.03 cells were treated with CCT137690 (C) or staurosporine (D) or erastin (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with CCT137690 (10 μM) for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F–I) Knockdown of RIPK1, RIPK3, and MLKL by shRNAs inhibited CCT137690 (10 μM, 24 hours)-induced cell death and HMGB1 and ATP release (n=3, *p < 0.05 versus control shRNA group).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Knockdown, Control, shRNA

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PDAC cells (n=3, *p < 0.05 versus control shRNA group). (C) Immunoprecipitation (IP) analysis of the levels of RIPK3 binding to RIPK1 and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (D) IP analysis of the levels of RIPK3 binding to RIPK1 and MLKL in control and stable AURKA-knockdown PANC1 and PANC2.03 cells. (E) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in PANC1 and PANC2.03 cells following treatment with CCT137690 (10 μM) for 24 hours. (F) IP analysis of the levels of AURKA binding to RIPK1, RIPK3, and MLKL in HEK293 cells after expressed AURKA wild type and D274A mutant. (G) PANC1 cells were treated with CCT137690 (10 μM) or TNF (50 ng/ml) in the absence or presence of anti-TNFR1 antibody (1 mg/ml) for 24 hours and cell death was analyzed (n=3, *p < 0.05).

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Control, Knockdown, shRNA, Immunoprecipitation, Binding Assay, Mutagenesis

Journal: Gastroenterology

Article Title: Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma

doi: 10.1053/j.gastro.2017.07.036

Figure Lengend Snippet: (A) Western blot analysis of indicated proteins in PDAC cells following treatment with CCT137690 (10 μM) for three to 24 hours (n=3, *p < 0.05 versus untreated group). (B) Western blot analysis of indicated proteins in control and stable AURKA-knockdown PANC1 and PANC2.03 cells (n=3, *p < 0.05 versus control shRNA group). (C, D) PANC1 and PANC2.03 cells were treated with XXVI (C) or AR-A014418 (D) in the absence or presence of indicated cell death inhibitors for 24 hours. Cell viability was assayed (n=3, *p < 0.05). (E) Indicated MEFs were treated with XXVI or AR-A014418 for 24 hours, and then cell viability was assayed (n=3, *p < 0.05). (F) Knockdown of RIPK3 and MLKL, but not RIPK1, inhibited XXVI- or AR-A014418-induced cell death (n=3, *p < 0.05 versus control shRNA group). (G–I) HEK293 cells were expressed with GSK3β WT and S9A mutant and then treated with CCT137690 (10 μM) for 24 hours. Protein level (G), cell viability (H), caspase-3 activity (H), and complex formation (I) were assayed.

Article Snippet: The antibodies to RIPK1 (#NB100-56160), LC3 (#NB100-2220), HMGB1 (#H00003146-M08), and AURKA (#NBP2-36737) were purchased from Novus Biologicals.

Techniques: Western Blot, Control, Knockdown, shRNA, Mutagenesis, Activity Assay