Journal: PLoS ONE

Article Title: Divergent Effects of Dioxin- or Non-Dioxin-Like Polychlorinated Biphenyls on the Apoptosis of Primary Cell Culture from the Mouse Pituitary Gland

doi: 10.1371/journal.pone.0146729

Figure Lengend Snippet: Representative Western blot from pituitary primary cell extracts obtained as described in the Materials and Methods. Primary pituitary cells were incubated for 24 hours with fresh serum-free medium containing the test substances at a 10 μM concentration or the vehicle (DMSO 0.1%). Cisplatin (32 μM) was used as a positive control. The expression of the final effector of apoptosis, i.e. caspase-3, changed in relation to the change in apoptosis during the exposure to a mixture of PCBs or individual congeners (A). In addition, the intrinsic (i.e., mithochondrial) apoptotic pathway was not influenced by either the mixture (Aroclor 1254) or the congener PCB 180, as shown by the unchanged expression of caspase-9 (C). In contrast, the non-dioxin-like PCB 180 and Aroclor 1254 increased the expression of caspase-8 (B), suggesting the involvement of the extrinsic pathway. Interestingly, the non-dioxin-like PCB 153 reduced the expression of both caspase-8 and 9 (B, C). Data are expressed as arbitrary units (A.U.), which represent the ratio between the intensity of the band of interest and the intensity of the band corresponding to the control protein (β-actin). The mean ± SD of measurements obtained in five independent experiments, are reported. *, p<0.01, **, p<0.001, ***, p<0.0001, compared with the vehicle, according to ANOVA followed by Dunnett’s post hoc test.

Article Snippet: Caspase activity was assessed by fluorimetric or luminometric assays using the NucView 488 Caspase-3 kit for live cells (Biotium, Hayward, CA, USA) and Caspase -8 or -9 Glo kit (Promega, Milan, Italy) respectively, as previously reported [ , ].

Techniques: Western Blot, Incubation, Concentration Assay, Positive Control, Expressing

Journal: PLoS ONE

Article Title: Divergent Effects of Dioxin- or Non-Dioxin-Like Polychlorinated Biphenyls on the Apoptosis of Primary Cell Culture from the Mouse Pituitary Gland

doi: 10.1371/journal.pone.0146729

Figure Lengend Snippet: Caspase activity was assessed by fluorimetric or luminometric assays, as described in the Materials and Methods. Cisplatin (32 μM) was included as a positive control. The activity of the three individual caspases decreased by exposure to PCB 153 (A, B, C). On the other hand, the activity of both caspase-3 and caspase-8 increased after treatment with Aroclor 1254 or PCB 180 (A, B), while the activity of caspase-9 was unaffected (C). To confirm the specificity of PCB action on caspases, cells were also pre-treated with caspase inhibitors prior to PCB exposure. 20 μM Ac-DEVD-CHO (A), Ac-IETD-CHO (B) or Ac-LEHD-CHO (C) were used as inhibitors of caspase -3, -8 or -9, respectively. Data (mean ± SD of five independent experiments) were expressed as arbitrary units (A.U.); the value of 1 was attributed to the caspase activity of vehicle-exposed cells. **, p<0.001, ***, p<0.0001, compared with the vehicle, according to ANOVA followed by Dunnett’s post hoc test. ##, p<0.005, ###, p<0.0005, p = n.s. (not significant), according to Student’s t-test adjusted for multiple comparisons following the Bonferroni method.

Article Snippet: Caspase activity was assessed by fluorimetric or luminometric assays using the NucView 488 Caspase-3 kit for live cells (Biotium, Hayward, CA, USA) and Caspase -8 or -9 Glo kit (Promega, Milan, Italy) respectively, as previously reported [ , ].

Techniques: Activity Assay, Positive Control

Journal: PLoS ONE

Article Title: Divergent Effects of Dioxin- or Non-Dioxin-Like Polychlorinated Biphenyls on the Apoptosis of Primary Cell Culture from the Mouse Pituitary Gland

doi: 10.1371/journal.pone.0146729

Figure Lengend Snippet: Quantitative ROS generation was measured using the oxidant-sensitive probe dichlorofluorescein (H2DCF-DA) assay (A), apoptosis was evaluated by ELISA DNA fragmentation assay (B), and caspase-3 activity was determined by fluorimetric assay (C), as described in the Materials and Methods. Pituitary primary cells were treated as described in the study design. 10 μM Hydrogen Peroxide (H 2 O 2 ) (A), 50 μM H 2 O 2 (B, C), or 32 μM Cisplatin (B,C) were used as positive controls. In all the assays, 1 mM Ascorbic Acid (vitamin C) was used as an antioxidant. ROS production was not influenced by non-dioxin-like PCB 180 or PCB 153, with or without the addition of vitamin C (A). Moreover, vitamin C had no effect on either apoptosis or caspase-3 activity of PCBs-treated pituitary cells (B, C), suggesting that ROS do not play a key role in PCBs-mediated apoptosis mechanism. Results represent the mean ± SD of five independent experiments, each performed in quadruplicate and expressed as arbitrary units (A.U.). The value of 1 was assigned to ROS production (A), DNA fragmentation (B) or caspase-3 activity (C) from vehicle-exposed cells. **, p<0.001, ***, p<0.0001 compared with vehicle, according to ANOVA followed by Dunnett’s post hoc test. #, p<0.05, ##, p<0.005, ###, p<0.0005, p = n.s. (not significant) according to Student’s t-test adjusted for multiple comparisons following the Bonferroni method.

Article Snippet: Caspase activity was assessed by fluorimetric or luminometric assays using the NucView 488 Caspase-3 kit for live cells (Biotium, Hayward, CA, USA) and Caspase -8 or -9 Glo kit (Promega, Milan, Italy) respectively, as previously reported [ , ].

Techniques: Enzyme-linked Immunosorbent Assay, DNA Fragmentation Assay, Activity Assay, Fluorimetry Assay

Journal: Autophagy

Article Title: Absence of autophagy promotes apoptosis by modulating the ROS-dependent RLR signaling pathway in classical swine fever virus-infected cells

doi: 10.1080/15548627.2016.1196318

Figure Lengend Snippet: Autophagy deficiency leads to the activation of apoptosis pathways in CSFV-infected cells. ((A)and C) PK-15 (A) and 3D4/2 (C) cells were transfected and infected as described in the legend to Fig. 2A and C. At 48 h after infection, the activity of CASP3, CASP8, and CASP9 in cells was measured by the CASP activity assay as described in Materials and Methods. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. *, P < 0.05; **, P < 0.01; #, P > 0.05. ((B)and D) PK-15 (B) and 3D4/2 (D) cells were transfected and infected as described in the legend to Fig. 2A and C. After 48 h, the expression of BCL2, BAX, cleaved-CASP3, cleaved-CASP8, cleaved-CASP9, cleaved-PARP, and ACTB (loading control) were analyzed by western blot with specific antibodies as described in Materials and Methods. The relative expression ratios of these proteins were analyzed by densitometric scanning. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. *, P < 0.05; ***, P < 0.001; #, P > 0.05.

Article Snippet: CASP activity assay The activation of CASP3, CASP8 and CASP9 in cells were measured using the CASP3/CPP32 Fluorometric Assay Kit (BioVision, K105-100), CASP8/FLICE Fluorometric Assay Kit (BioVision, K112-100), and CASP9 Fluorometric Assay Kit (BioVision, K118-100) following the manufacturer's instructions, respectively.

Techniques: Activation Assay, Infection, Transfection, Activity Assay, Expressing, Western Blot

Journal: Autophagy

Article Title: Absence of autophagy promotes apoptosis by modulating the ROS-dependent RLR signaling pathway in classical swine fever virus-infected cells

doi: 10.1080/15548627.2016.1196318

Figure Lengend Snippet: Type I IFN production is required for the activation of the extrinsic apoptosis pathway. (A) PK-15 and 3D4/2 cells were transfected and infected as described in the legend to Fig. 2A and C, which were considered as the control groups. For inhibition of IFNA and IFNB1 induction, cells were cultured with bufexamac (10 μM) after 1 h of virus absorption, or cotransfected with shRNA targeting IFNB1 prior to CSFV infection. At 48 h after infection, the mRNA levels of TNFSF10, TNFRSF10A, FASLG, and FAS were measured by qRT-PCR. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P > 0.05. (B) PK-15 and 3D4/2 cells were treated as described in (A). After 48 h, the activity of CASP3, CASP8, and CASP9 in cultured cells was detected by the CASP activity assay as described in Materials and Methods. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. *, P < 0.05; **, P < 0.01; #, P > 0.05. (C) PK-15 and 3D4/2 cells were treated as described in (A). After 48 h, cell samples were analyzed by western blot with antibodies against PARP and ACTB (loading control). The relative expression ratios of the targeted proteins were analyzed by densitometric scanning. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. ***, P < 0.001. (D) PK-15 and 3D4/2 cells were treated as described in (A). After 48 h, IFNA and IFNB1 production and the inhibition efficiency of type I IFN induction were detected by the ELISA assay. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P > 0.05.

Article Snippet: CASP activity assay The activation of CASP3, CASP8 and CASP9 in cells were measured using the CASP3/CPP32 Fluorometric Assay Kit (BioVision, K105-100), CASP8/FLICE Fluorometric Assay Kit (BioVision, K112-100), and CASP9 Fluorometric Assay Kit (BioVision, K118-100) following the manufacturer's instructions, respectively.

Techniques: Activation Assay, Transfection, Infection, Inhibition, Cell Culture, shRNA, Quantitative RT-PCR, Activity Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: Absence of autophagy promotes apoptosis by modulating the ROS-dependent RLR signaling pathway in classical swine fever virus-infected cells

doi: 10.1080/15548627.2016.1196318

Figure Lengend Snippet: Regulation of ROS levels within autophagy-impaired cells triggers the extrinsic apoptosis pathway during CSFV infection. (A) PK-15 and 3D4/2 cells were treated and cultured as described in the legend to Fig. 9A. The activities of CASP3, CASP8, and CASP9 in cell samples were assessed by the CASP activity assay as described in Materials and Methods. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P < 0.05, Dunnett's T3 (3) was used for correction of post-hoc test. *, P < 0.05; **, P < 0.01; #, P > 0.05. (B) PK-15 and 3D4/2 cells were treated and cultured as described in the legend to Fig. 10A and B. The levels of cleaved-PARP and ACTB (loading control) were detected by western blot using specific antibodies. The relative expression ratios of these proteins were assessed by densitometric scanning. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) PK-15 and 3D4/2 cells were transfected as described in the legend to Fig. 9A. At 24 and 48 h after CSFV infection, virus copy number was detected by qRT-PCR as described in Materials and Methods. The data represent the mean ± SD of 3 independent experiments. One-way ANOVA test; test of homogeneity of variances, P > 0.05, LSD (L) was used for correction of post-hoc test. **, P < 0.01; ***, P < 0.001.

Article Snippet: CASP activity assay The activation of CASP3, CASP8 and CASP9 in cells were measured using the CASP3/CPP32 Fluorometric Assay Kit (BioVision, K105-100), CASP8/FLICE Fluorometric Assay Kit (BioVision, K112-100), and CASP9 Fluorometric Assay Kit (BioVision, K118-100) following the manufacturer's instructions, respectively.

Techniques: Infection, Cell Culture, Activity Assay, Western Blot, Expressing, Transfection, Quantitative RT-PCR

Journal: Molecular Metabolism

Article Title: Dual specificity phosphatase 1 attenuates inflammation-induced cardiomyopathy by improving mitophagy and mitochondrial metabolism

doi: 10.1016/j.molmet.2022.101567

Figure Lengend Snippet: DUSP1 attenuates the inflammatory response and cardiomyocyte death. DUSP1 TG and WT mice were injected intraperitoneally with a single dose of lipopolysaccharide (20 mg/kg) to induce SCM, and were evaluated after 48 h. HL-1 cells were treated with 10 μg/mL lipopolysaccharide for 24 h to induce inflammatory cardiomyocyte damage. HL-1 cells were transfected with the DUSP1-LV to overexpress DUSP1 before lipopolysaccharide treatment. A-C. RNA was collected and used for qPCR. The relative levels of IL-6 , TNFα and MCP1 were normalized to GAPDH levels. D, E . Immunofluorescence staining was used to assess the expression of Gr1, a neutrophil cell-surface marker. TnT was used to label the myocardium. F, G. Immunohistochemistry was applied to observe changes in ICAM1 expression in the myocardium. H. An ELISA was used to measure caspase-3 activity in heart tissues. I, J. TUNEL staining was applied to observe the number of apoptotic cardiomyocytes. K. An MTT assay was used to determine the viability of cardiomyocytes in the presence of lipopolysaccharide. ∗p < 0.05.

Article Snippet: Caspase-3 activity was measured with a Caspase-3 Activity Assay Kit (fluorometric, #ab252897, Abcam).

Techniques: Injection, Transfection, Immunofluorescence, Staining, Expressing, Marker, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Activity Assay, TUNEL Assay, MTT Assay

Journal: Molecular Metabolism

Article Title: Dual specificity phosphatase 1 attenuates inflammation-induced cardiomyopathy by improving mitophagy and mitochondrial metabolism

doi: 10.1016/j.molmet.2022.101567

Figure Lengend Snippet: FUNDC1 deficiency abolishes the beneficial effects of DUSP1 on cardiomyocyte mitochondria. DUSP1 TG and WT mice were injected intraperitoneally with a single dose of lipopolysaccharide (20 mg/kg) to induce SCM, and were evaluated after 48 h. HL-1 cells were treated with 10 μg/mL lipopolysaccharide for 24 h to induce inflammatory cardiomyocyte damage. HL-1 cells were transfected with the DUSP1-LV to overexpress DUSP1 before lipopolysaccharide treatment. HL-1 cells were transfected with shRNA against FUNDC1 (sh-FUNDC1) before DUSP1-LV transfection. A-E. The OCR was used to analyze mitochondrial respiration in the presence of lipopolysaccharide. State-3/4 mitochondrial respiration was recorded, along with the RCR, ADP/O and lag phase. F, G. Mitochondrial ROS production was determined via immunofluorescence. H. An MTT assay was used to determine cell viability. I. An ELISA was used to determine caspase-3 activity. ∗p < 0.05.

Article Snippet: Caspase-3 activity was measured with a Caspase-3 Activity Assay Kit (fluorometric, #ab252897, Abcam).

Techniques: Injection, Transfection, shRNA, Immunofluorescence, MTT Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: MPTP mediated Ox-mtDNA release inducing macrophage pyroptosis and exacerbating MCD-induced MASH via promoting the ITPR3/Ca 2+ /NLRP3 pathway

doi: 10.1186/s12967-025-07302-8

Figure Lengend Snippet: Macrophages in MCD animal model showed significant pyroptosis and Ox-mtDNA release. Liver in mice were subjected to MCD or normal diet for 4 weeks: A. Schematic diagram of the animal experiment modeling process. B-C . GDSMD and Caspase1 were detected using IHC in liver tissue sections (scale bar, 50µM) and area (%) of protein expression. D-E . Liver macrophages in each group were isolated, and the levels of Caspase1, cleaved-Caspase1, GSDMD and GSDMD-N were measured by WB. F . Liver macrophages in each group were isolated, and Caspase1 activity was determined with the Caspase1 assay kit. G . Serum levels of LDH were measured. H . The levels of serum inflammatory factors (IL-1β and IL-18) were tested by ELISA. I-J. TEM was used to observe the ultrastructural changes in macrophages (scale bar, 2µM and 100 nm) and mitochondrial swelling score. K . 8-OH-dG from total cell was quantified using the 8-OH-dG ELISA kit. L . 8-OH-dG from cytosol was quantified using the 8-OH-dG ELISA kit. liver macrophages from normal mice and liver macrophages from MCD animal model were measured. M . Relative cytosolic mtDNA amounts in each group. The relative ratios of D-loop mtDNA, COX1 mtDNA, or Non-Numt mtDNA are tested by qPCR. All data are shown as the mean ± SD ( n = 6). *** P < 0.001, ** P < 0.01 and * P < 0.05

Article Snippet: Caspase1 enzymatic activity was assessed in both primary liver macrophages and THP-1 derived macrophages using a fluorometric Caspase1 activity assay Kit (Beyotime, C1101), with all procedures performed in strict accordance with the manufacturer’s instructions.

Techniques: Animal Model, Expressing, Isolation, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: MPTP mediated Ox-mtDNA release inducing macrophage pyroptosis and exacerbating MCD-induced MASH via promoting the ITPR3/Ca 2+ /NLRP3 pathway

doi: 10.1186/s12967-025-07302-8

Figure Lengend Snippet: Blocking Ox-mtDNA release mitigates pyroptosis of macrophages and liver damage in MCD animal model. Mice were fed CsA10 mg/kg/day continuously for 7 weeks, and the MCD diet was started in the third week. Isolate each group of liver macrophages: ( A ) Schematic diagram of the feeding process of mice in each group. ( B ) 8-OH-dG in cytosol were quantified using ELISA. ( C ) Relative cytosolic mtDNA amounts in each group. The relative ratios of D-loop mtDNA, COX1 mtDNA, or Non–Numt mtDNA are tested by qPCR. D - E . The levels of Caspase1, cleaved-Caspase1, GSDMD and GSDMD-N were measured by WB. F . Caspase1 activity was determined with the Caspase1 assay kit. G . Supernatant levels of LDH were measured by LDH assay kit. H . Supernatant levels of IL-1β and IL-18 were tested by ELISA. I - J . NAS scores are based on H&E-stained liver sections (scale bars, 100µM) from different groups of mice. K - L . Oil red staining is based on liver sections (scale bar, 100µM) from different groups of mice and relative area of lipid droplets. M - N . Serum levels of ALT, AST and liver TG were measured by biochemical kit. All data are shown as the mean ± SD ( n = 6). *** P < 0.001, ** P < 0.01 and * P < 0.05

Article Snippet: Caspase1 enzymatic activity was assessed in both primary liver macrophages and THP-1 derived macrophages using a fluorometric Caspase1 activity assay Kit (Beyotime, C1101), with all procedures performed in strict accordance with the manufacturer’s instructions.

Techniques: Blocking Assay, Animal Model, Enzyme-linked Immunosorbent Assay, Activity Assay, Lactate Dehydrogenase Assay, Staining

Journal: Journal of Translational Medicine

Article Title: MPTP mediated Ox-mtDNA release inducing macrophage pyroptosis and exacerbating MCD-induced MASH via promoting the ITPR3/Ca 2+ /NLRP3 pathway

doi: 10.1186/s12967-025-07302-8

Figure Lengend Snippet: FFA induces Ox-mtDNA release via mPTP, leading to pyroptosis of THP-1 derived macrophages. THP-1 derived macrophages were cultured for 24 h with normal or FFA added. ( A ) Representative of immunofluorescence staining of Calcein (green) and corresponding cell density (white light). ( B ) 8-OH-dG in cytosol were quantified using ELISA. ( C ) Relative cytosolic mtDNA amounts in each group. The relative ratios of D-loopHV1 mtDNA, COX2 mtDNA, or ND1 mtDNA are tested by qPCR. ( D ) The colocalization of Mito-mCherry (red) and dsDNA (green) was detected by confocal microscopy. E - F . The levels of Caspase1, cleaved-Caspase1, GSDMD and GSDMD-N were measured by WB. G . Caspase1 activity was determined with the Caspase1 assay kit. H . Supernatant levels of LDH were measured by LDH assay kit. I . Supernatant levels of IL-1β and IL-18 were tested by ELISA. J-K . TEM was used to observe the ultrastructural changes in macrophages (scale bar, 2µM and 100 nm) and mitochondrial swelling score. All data are shown as the mean ± SD ( n = 6). *** P < 0.001, ** P < 0.01 and * P < 0.05

Article Snippet: Caspase1 enzymatic activity was assessed in both primary liver macrophages and THP-1 derived macrophages using a fluorometric Caspase1 activity assay Kit (Beyotime, C1101), with all procedures performed in strict accordance with the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Activity Assay, Lactate Dehydrogenase Assay

Journal: Journal of Translational Medicine

Article Title: MPTP mediated Ox-mtDNA release inducing macrophage pyroptosis and exacerbating MCD-induced MASH via promoting the ITPR3/Ca 2+ /NLRP3 pathway

doi: 10.1186/s12967-025-07302-8

Figure Lengend Snippet: Blocking Ox-mtDNA release attenuates THP-1 derived macrophages pyroptosis and reduces lipid droplets in vitro. In the absence or presence of CsA (1µM), THP-1 derived macrophages were cultured normally as well as FFA/DMSO added. ( A ) Representative of immunofluorescence staining of Calcein (green) and corresponding cell density (white light). ( B ) 8-OH-dG in cytosol were quantified using ELISA. ( C ) Relative cytosolic mtDNA amounts in each group. The relative ratios of D-loopHV1 mtDNA, COX2 mtDNA, or ND1 mtDNA are tested by qPCR. ( D ) The colocalization of Mito-mCherry (red) and dsDNA (green) was detected by confocal microscopy. E-F. The levels of Caspase1, cleaved-Caspase1, GSDMD and GSDMD-N were measured by WB. G . Caspase1 activity was determined with the Caspase1 assay kit. H . Supernatant levels of LDH were measured by LDH assay kit. I . Supernatant levels of IL-1β and IL-18 were tested by ELISA. J - K . TEM was used to observe the ultrastructural changes in macrophages (scale bar, 2µM) and mitochondrial swelling score. L - M . Oil red staining is based on each group of cells and relative area of lipid droplets. All data are shown as the mean ± SD ( n = 6). *** P < 0.001, ** P < 0.01 and * P < 0.05

Article Snippet: Caspase1 enzymatic activity was assessed in both primary liver macrophages and THP-1 derived macrophages using a fluorometric Caspase1 activity assay Kit (Beyotime, C1101), with all procedures performed in strict accordance with the manufacturer’s instructions.

Techniques: Blocking Assay, Derivative Assay, In Vitro, Cell Culture, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Activity Assay, Lactate Dehydrogenase Assay

Journal: Journal of Translational Medicine

Article Title: MPTP mediated Ox-mtDNA release inducing macrophage pyroptosis and exacerbating MCD-induced MASH via promoting the ITPR3/Ca 2+ /NLRP3 pathway

doi: 10.1186/s12967-025-07302-8

Figure Lengend Snippet: Inhibition of ITPR3/Ca²⁺ axis alleviates NLRP3-dependent pyroptosis in liver macrophages and reduces the intracellular lipid droplets. Treat THP-1 derived macrophages with si-ITPR3/Scramble/Iono, cultured with normal or FFA for 24 h: A . The Fluo4 series calcium fluorescence indicator kit measures the concentration of Ca 2+ in the cytosol and the corresponding cell density (white light). B - E . The levels of ITPR3, NLRP3, Caspase-1, cleaved-Caspase1, GSDMD and GSDMD-N were measured by WB. F . Caspase1 activity was determined with the Caspase1 assay kit. G . Supernatant levels of LDH were measured by LDH assay kit. H . Supernatant levels of IL-1β and IL-18 were tested by ELISA. I - J . TEM was used to observe the ultrastructural changes in macrophages (scale bar, 2µM) and mitochondrial swelling score. K - L . Oil red staining is based on each group of cells and relative area of lipid droplets. All data are shown as the mean ± SD ( n = 6). *** P < 0.001, ** P < 0.01 and * P < 0.05

Article Snippet: Caspase1 enzymatic activity was assessed in both primary liver macrophages and THP-1 derived macrophages using a fluorometric Caspase1 activity assay Kit (Beyotime, C1101), with all procedures performed in strict accordance with the manufacturer’s instructions.

Techniques: Inhibition, Derivative Assay, Cell Culture, Fluorescence, Concentration Assay, Activity Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Journal of Translational Medicine

Article Title: MPTP mediated Ox-mtDNA release inducing macrophage pyroptosis and exacerbating MCD-induced MASH via promoting the ITPR3/Ca 2+ /NLRP3 pathway

doi: 10.1186/s12967-025-07302-8

Figure Lengend Snippet: Inhibition of ITPR3/Ca²⁺ signaling blocks Ox-mtDNA induced liver macrophages pyroptosis and mitigates MCD animal model damage. The mice in the experimental group were injected with ITPR3-AAV through the tail vein: A . Schematic diagram of animal experiments. B - E . The levels of ITPR3, NLRP3, Caspase-1, cleaved-Caspase1, GSDMD and GSDMD-N were measured by WB. F . Caspase1 activity was determined with the Caspase1 assay kit. G . Supernatant levels of LDH were measured by LDH assay kit. H . Supernatant levels of IL-1β and IL-18 were tested by ELISA. I - J . NAS score and lipid droplets relative area analysis were based on liver slice H&E staining and oil red staining (scale bar, 100µM) of different groups of mice. K - L . Serum levels of ALT, AST and liver TG were measured by biochemical kit. All data are shown as the mean ± SD ( n = 6). *** P < 0.001, ** P < 0.01 and * P < 0.05

Article Snippet: Caspase1 enzymatic activity was assessed in both primary liver macrophages and THP-1 derived macrophages using a fluorometric Caspase1 activity assay Kit (Beyotime, C1101), with all procedures performed in strict accordance with the manufacturer’s instructions.

Techniques: Inhibition, Animal Model, Injection, Activity Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Staining