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