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The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and <t>CRISPR-Caspase</t> 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, <t>p-RIPK1,</t> <t>GSDMD-N,</t> cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.
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The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and <t>CRISPR-Caspase</t> 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, <t>p-RIPK1,</t> <t>GSDMD-N,</t> cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.
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The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and <t>CRISPR-Caspase</t> 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, <t>p-RIPK1,</t> <t>GSDMD-N,</t> cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.
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The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and <t>CRISPR-Caspase</t> 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, <t>p-RIPK1,</t> <t>GSDMD-N,</t> cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.
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The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and <t>CRISPR-Caspase</t> 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, <t>p-RIPK1,</t> <t>GSDMD-N,</t> cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.
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crispr cas9 knockout expression vector  (Santa Cruz Biotechnology)
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The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and <t>CRISPR-Caspase</t> 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, <t>p-RIPK1,</t> <t>GSDMD-N,</t> cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.
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The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and CRISPR-Caspase 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, p-RIPK1, GSDMD-N, cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.

Journal: Hepatology Communications

Article Title: NUAK1 promotes metabolic dysfunction-associated steatohepatitis progression by activating Caspase 6–driven pyroptosis and inflammation

doi: 10.1097/HC9.0000000000000479

Figure Lengend Snippet: The NUAK1/Caspase 6 signaling is required for the crosstalk between TAK1 and RIPK1 in MASH-induced hepatocytes. Mouse hepatocytes were treated with HTH-01-015 and CRISPR-Caspase 6 activation vectors, followed by PA stimulation. (A) Oil Red O staining of PA-stimulated hepatocytes (scale bar, 50 and 20 μm). (B) LDH releasing from hepatocytes. (C) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in hepatocytes. (D) ELISA analysis of IL-1β levels in culture medium from PA-stimulated hepatocytes. (E) Caspase 6 activity in hepatocytes. (F) Western blot analysis and relative density ratio of aCaspase 6, TAK1, RIPK1, p-RIPK1, GSDMD-N, cCaspase 1, and NLRP3 in PA-stimulated hepatocytes. (G) Immunofluorescence staining of TAK1 (red) and RIPK1 (green) in hepatocytes after treatment with HTH-01-015. DAPI was used to visualize nuclei (blue) (scale bars, 20 and 10 μm). (H) Immunoprecipitation analysis of TAK1 and RIPK1 in hepatocytes induced by PA. ANOVA, Bonferroni multiple comparison test, n=4–6 per group. All data represent the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: LDH, lactate dehydrogenase; PA, palmitic acid; qRT-PCR, quantitative real time-PCR.

Article Snippet: The AML12 strain of mouse hepatocytes (1 × 10 6 ) was transfected with CRISPR/Cas9 Caspase 6 KO, TAK1 KO, RIPK1 KO, GSDMD KO, CRISPR Caspase 6, TAK1, RIPK1, or GSDMD activation, and CRISPR control vector (Santa Cruz) through using Lipofectamine 3000 reagent (L3000075, Invitrogen) in antibiotic-free medium.

Techniques: CRISPR, Activation Assay, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot, Immunofluorescence, Immunoprecipitation, Comparison, Real-time Polymerase Chain Reaction