Fig. 6.

Synthetic ss-miR mimics for plant miR159a and miR156c target the Tnf signaling pathway. a, b Tnfrsf1a protein levels in NV-treated adipocytes (a) and vWAT (b) of mice fed with high-fat diet (HFD) or HFD supplemented with NVs (HFD + NV). Uncropped images are shown in Supplementary Fig. 9. c, d Tnfrsf1a and p-NFkBp65 protein levels (c) and TNF-α mRNA expression (d) were analyzed in hypertrophic 3T3-L1 adipocytes transfected with single-stranded (ss) 2′-O-methylated miR159a or miR156c mimics. Transfection with ss-2′-O-methylated miR167h mimic or with a scramble small RNA [(−)sRNA] was used as negative control. Uncropped images are shown in Supplementary Fig. 9. e–g p-NFkBp65 and/or Tnfrsf1a protein levels were analyzed in differentiated T37i brown adipocytes (e), in primary human monocytes differentiated in M1 macrophages (f) and TNF-α-treated murine RAW 264.7 cells (g) transfected with ss-2′-O-methylated miR159a or miR156c mimics. Transfection with a scramble small RNA [(−)sRNA] was used as a negative control. Uncropped images are shown in Supplementary Fig. 9. h, i Tnfrsf1a protein levels (h) and TNF-α mRNA expression (i) were analyzed in T37i brown adipocytes transfected with single-stranded (ss) 2′-O-methylated, unmethylated miR156c, or miR159a mimics. Transfection with a scramble small RNA [(−)sRNA] was used as a negative control. Immunoblots reported are representative of three independent experiments giving similar results. Tubulin, actin, Hsp60, or Tom20 were used as loading controls. Uncropped images are shown in Supplementary Fig. 9. Data are expressed as means ± SD (n = 3). j HEK293 cells were transfected with the reporter construct (pGL3–5′Tnfrsf1a-LUC), containing the mouse 5′Tnfrsf1a region with predicted miR-binding sites (left panel) cloned upstream the luciferase gene, together with ss-2′-O-methylated miR159a, ss-2′-O-methylated miR156c mimics or negative control (ss-2′-O-methylated miR167h mimic). Firefly/Renilla luciferase activities were averaged (n = 10) and reported as residual activity of the respective transfections performed with negative control taken as 100%