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. 2014 Dec 24;43(2):1177–1188. doi: 10.1093/nar/gku1343

Figure 4.

Figure 4.

Inhibition of RNA sensing by 2′OMe is motif dependent. (A) BMMs from WT mice pre-treated with the indicated dose of AMO or RD for 45 min were stimulated overnight with 180 nM of B-406AS-1, and TNF-α levels were measured in supernatants by ELISA. The data are averaged from three independent experiments in biological triplicate. Percentages of cytokine production compared to the 0.6 nM RD+B-406AS-1 condition are given. Ordinary two-way ANOVA with Dunnett's multiple comparison tests to miR-200c-3p+B-406AS-1 are shown. (B) Human PBMCs were pre-treated for 45 min with the indicated dose of AMO or RD, and stimulated overnight with 180 nM of B-406AS-1. TNF-α levels were measured in supernatants by ELISA. The data are averaged from three independent experiments in different blood donors, in biological triplicate. Unpaired t-tests comparing miR-200a-3p+B-406AS-1 and indicated conditions are shown. (C) Top: alignment of miR-200a/b/c-3p AMOs with the core inhibitory motif highlighted in grey. Bottom: MEME pictogram of the relative frequency of bases constituting the inhibitory motif (40). (D) Alignment of miR-200a-3p, miR-200c-3p, miR-122-5p and NC1 AMO variants with altered/truncated inhibitory motifs (in 5′-3′ orientation). Native inhibitory motifs are underlined and point mutations are in red bold. BMMs from WT mice (E, F, G, I) or human PBMCs (F, H) pre-treated with 40 nM of the indicated 2′OMe AMO or RD were stimulated overnight with 180 nM of B-406AS-1, and TNF-α levels were measured in supernatants by ELISA. Percentages of cytokine production compared to the RD+B-406AS-1 condition are given. The data are averaged from a minimum of two independent experiments, in biological triplicate. Ordinary one-way ANOVA with Dunnett's multiple comparison tests to miR-200a-3p+B-406AS-1 (E) or NC1+B-406AS-1 (H and I) are shown. Unpaired t-tests comparing miR-200a-3p+B-406AS-1 (F) or RD+B-406AS-1 (G) and indicated conditions are shown (ns, not significant). (A, E, H, I) Unless otherwise indicated, differences were not significant. (A, B and E–I) SEM is shown.