Primer sequences for genes measured with qPCR.

Hepatic lipid levels and the expression of lipid uptake receptors are increased by a whole-body knockout of miR-26b in mice. (A) Schematic overview of the experimental approach. (B-C) Hepatic total cholesterol (B) and triglyceride (C) measurements normalized against total protein. (D) Representative pictures of Oil-red-O staining of liver sections. Scale bar=200µm. (E) Quantification of the Oil-red-O staining. (F) Pathological scoring of the Oil-red-O staining. (G-J) Gene expression analysis of (G) Abca1, (H) Acat2, (I) Cd36, and (J) Sra. Fold change is corrected for sex. *p<0.05; **p<0.01. n=6-7 animals per group.

Hepatic lipid levels and the expression of lipid uptake receptors are increased by a myeloid-specific miR-26b deficiency in mice.

(A) Schematic overview of the experimental approach. (B-C) Hepatic total cholesterol (B) and triglyceride (C) measurements normalized against total protein. (D) Quantification of the Oil-red-O staining. (E-F) Gene expression analysis of (E) Cd36 and (F) Sra. Fold change is corrected for sex. *p<0.05; **p<0.01. n=6-8 animals per group.

Livers of Apoe-/-Mir26b-/-mice show elevated pro-inflammatory cytokine levels and an increased number of Mac-1-positive cells.

(A-D) Cytokine levels of (A) IL-6, (B) TNF, (C) CCL2, and (D) CXCL1 were measured in liver protein lysates. (E-H) Representative images and quantification of immunofluorescent stainings for (E) infiltrating macrophages and neutrophils (Mac-1), (F) neutrophils (Ly6G), (G) resident monocytes/macrophages (CD68), and (H) T-cells (CD3). Scale bar=50μm. *p<0.05; **p<0.01. n=6-7 animals per group.

Hepatic fibrosis is exacerbated by a whole-body knockout of miR-26b in mice.

(A) Representative pictures of Sirius-red staining of liver sections. Scale bar=100µm. (B) Quantification of the Sirius-red staining. (C-E) Gene expression of (C) Tgfb, (D) Acta2, and (E) Mmp13. Fold change is corrected for sex. *p<0.05. n=6-7 animals per group.

Knockout of miR-26b results in an increased hepatic inflammatory kinase activity.

(A) Principal component analysis (PCA) of phosphorylated peptides from STK array (n=4) of liver lysates from Apoe-/-Mir26b-/- mice (KO) or Apoe-/- (WT) mice. (B) Volcano plot visualizing fold change and p-value for phosphorylated peptides from STK array. Blue dots represent significantly altered phosphopeptides. (C) Heatmap of significantly changed kinases are ranked based on Median Final Score (cut-off value of 1.2), STK array performed on liver lysates from Apoe-/-Mir26b-/- mice (KO) compared to Apoe-/- (WT) mice. Color corresponds to the Median Kinase Statistic, which represents effect size and directionality (red = increased activity in KO vs. WT mice). (D) Enriched Pathways based on STK array. (E) Network diagram of the pathway enrichment analysis.

Apoe-/-Mir26b-/-mice injected with LNPs containing miR-26b mimics show decreased hepatic lipid levels compared to vehicle control injected mice.

(A) Schematic overview of the experimental approach in which mice on 4-week WTD were simultaneously injected every 3 days with either empty LNPs as vehicle control (eLNP) or LNPs containing miR-26b mimics (mLNP). (B-C) Hepatic total cholesterol (B) and triglyceride (C) measurements normalized against total protein. (D) Quantification of the Oil-red-O staining. (E-F) Gene expression analysis of (E) Cd36 and (F) Sra. Fold change is corrected for sex. *p<0.05; **p<0.01. n=6 animals per group.

mLNP treatment of miR-26b knockout mice results in a decreased hepatic inflammatory kinase activity.

(A) Principal component analysis (PCA) of phosphorylated peptides from STK array (n=4) of liver lysates from mLNP treated Apoe-/-Mir26b-/- mice (KO.LNP), Apoe-/-Mir26b-/- mice (KO) or Apoe-/- mice (WT) mice. (B) Volcano plot visualizing fold change and P value for phosphorylated peptides from STK array. Blue dots represent significantly altered phosphopeptides. (C) The heatmap of significantly changed kinases is ranked based on the Median Final Score (cut-off value of 1.2). Color is corresponding to Median Kinase Statistic, which represents effect size and directionality (red = increased activity in KO vs. WT mice; blue = decreased activity in KO.LNP vs. KO mice) (average of n=4 is shown). (D) Enriched Pathways based on STK array. (E) Network diagram of the pathway enrichment analysis.

MiR-26b-loaded LNPs have anti-inflammatory effects on human liver slices and miR-26b plasma levels are significantly increased in patients with liver cirrhosis.

(A) Schematic overview of the experimental approach. (B-E) Cytokine levels of (B) IL-6, (C) TNF, (D) CCL2, and (E) CXCL1 measured in the supernatant of human precision-cut liver slices after 24h (for IL-6/TNF) or 48h (for CCL2/CXCL1) incubation with mLNPs or eLNPs (n=3). (F-G) Plasma was isolated from patients with liver cirrhosis or healthy volunteers (F) and miR-26b-3p (G) and miR-26b-5p (H) plasma levels were measured. *p<0.05; ****p<0.0001. n=8-11 patients per group.