Fig 2: NUAK1 inhibition suppressed Caspase 6 activity and induced TAK1 activation and cell death in mouse MASH models. Four-week-old mice were fed with HFD for 24 weeks, accompanied by the treatment of NUAK1 inhibitor HTH-01-015. (A) Western blot analysis and relative density ratio of active Caspase 6, TAK1, RIPK1, p-RIPK1, GSDMD-N, cCaspase 1, and NLRP3. (B) Immunohistochemistry staining of p-RIPK1 in fatty livers (scale bars, 50 μm). (C) Caspase 6 activity in the liver lysate. (D) qRT-PCR analysis of IL-1β, IL-6, TNF-α, and TGF-β in fatty livers. (E) ELISA analysis of serum IL-1β levels. (F) ELISA analysis of serum IL-18 levels. 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: HFD, high-fat diet; qRT-PCR, quantitative real time-PCR.

Fig 3: 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.

Fig 4: NUAK1 is upregulated in the livers of patients with MASH or mouse MASH models. (A) NUAK1 mRNA in the liver tissues of human MASLD (NAS <5) and MASH (NAS ≥5), compared with normal individuals, as determined by qRT-PCR (n = 18 per group). (B) NUAK1 protein levels in the liver tissues of human normal individuals and MASH, as determined by western blot (n = 5 per group). (C) NUAK1 immunohistochemistry staining in liver specimens of patients with MASH and normal individuals (scale bar: 50 µm). (D) Correlation between NUAK1 mRNA and liver TGF-β expression in human MASH livers. (E–G) mRNA and protein levels of NUAK1 were detected in the livers of mice fed with HFD or MCD compared with NCD. (H) Double immunofluorescence staining of NUAK1 (green) and HNF-4α (red) in liver samples of mice fed on NCD or HFD (scale bar, 50 μm). DAPI was used to visualize nuclei (blue). 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: HFD, high-fat diet; MCD; methionine-choline–deficient diet; NAS, nonalcoholic fatty liver disease activity score; NCD, normal chow diet; qRT-PCR, quantitative real time-PCR.