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. 2023 Oct 11;14:6384. doi: 10.1038/s41467-023-42040-9

Fig. 6. Trim26 alleviates NASH by suppressing the Cebpd-Hif1a pathway cascade.

Fig. 6

a Representative western blotting bands showing the CCAAT/enhancer binding protein delta (Cebpd), hypoxia-inducible factor-1a (Hif1a), nitric oxide synthase 2 (Nos2), phosphorylated and total p38 and NF-κB p65 protein expression in livers of indicated groups (n = 4 per group). b Representative western blotting bands showing the Cebpd, Hif1a, Nos2, phosphorylated and total p38 and NF-κB p65 protein expression in livers of indicated groups (n = 4 per group). c Representative western blotting bands displaying the CEBPD, HIF1A, NOS2, phosphorylated and total P38 and P65 protein expression in indicated groups (n = 4 per group). d Representative immunoblotting bands displaying the CEBPD, HIF1A, NOS2, phosphorylated and total P38 and P65 protein expression in indicated groups (n = 4 per group). e Representative Nile red staining images displaying the lipid deposition after 10 h, 0.5 mM palmitate+1.0 mM oleic acid (PAOA)-incubated transfected or co-transfected cultured L02 cells (upper; P < 0.0001 by one-way ANOVA) and mice primary hepatocytes (lower; P < 0.0001 by one-way ANOVA) (scale bars: magnification, 100×; n = 10 per group). f Co-immunoprecipitation detection of the interaction of TRIM26 with CEBPD in L02 cells transfected with TRIM26-Flag or CEBPD-HA plasmids. Immunoblotting probed with anti-HA or anti-Flag antibody (upper). Representative immunoblotting bands for GST precipitation showing TRIM26-CEBPD binding by treating purified CEBPD-His with purified TRIM26-HA-GST or by treating TRIM26-His with purified CEBPD-HA-GST in vitro (lower). Purified GST was regarded as a control. g Schematic of human full-length and truncated TRIM26 and CEBPD (upper), and representative western blotting mapping assay indicating the interaction domains of TRIM26 and CEBPD (lower). h TRIM26 structure showing the two conservative motifs (TRIM26(CXXC), Motif-1# & TRIM26(CXXXXC), Motif-2#) of RING domain in different species. i Representative Nile red staining images (P < 0.0001 by one-way ANOVA) and intracellular TG levels (P < 0.0001 by one-way ANOVA) showing the lipid accumulation in PAOA-treated AdGFP-, AdTRIM26(AXXA)-, AdTRIM26(AXXXXA)- or AdTRIM26-transfected L02 cells for 10 h. The L02 cells transfected with AdGFP were used as a control (scale bars: magnification, ×100; n = 10 per group). j Representative western blotting bands displaying the TRIM26, CEBPD, HIF1A, NOS2, phosphorylated and total P38 and P65 protein expression in PAOA-treated transfected L02 cells (n = 4 per group). k qPCR assay showing the inflammation- and fatty acid synthesis-associated genes expression alteration in PAOA-treated transfected L02 cells (n = 4 per group; P < 0.05 by one-way ANOVA). Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. P < 0.05 indicates statistical significance.