Supplementary Figure 4

(a) Sequences of mmu-miR-193b and mmu-miR-328, comparison of miR-193b versus miR-328 seed sequences (top) and overlap of in silico predicted miR-193b (grey) and miR-328 mRNA (black) targets (bottom). The statistical significance for the overlap of miR-193b and miR-328 target genesets is given in Methods. (b) Relative luciferase activity in HEK293 cells after co-transfection of pmiRGlo reporter constructs harbouring wild-type 3′UTR fragments of indicated genes together with 100 nM of scr, miR-193b or miR-328 mimics across n = 3 independent experiments. (c) Relative expression of Bace1 (n = 6) and Gprc5b (n = 8) in indicated tissues (BAT, brown adipose tissue; H, heart; Liv, liver; SM, skeletal muscle; WAT, white adipose tissue). Expression levels in BAT were set to unity = 1. (d) Relative expression of Bace1 and Gprc5b in C2C12 cells seven days after transfection with 80 nM of indicated longRNA GapmeRs across n = 4 (Bace1) or n = 5 (Gprc5b) independent experiments, left). Relative expression of Bace1 in PIBA cells two days after transfection with 80 nM of indicated longRNA GapmeRs across n = 3 independent experiments, right). (e) Photomicrograph of C2C12 cells seven days after transfection with 80 nM of indicated longRNA Gapmers. Image is representative of three independent experiments. Scale bar, 100 μm. (f) Relative expression of Myf5, Myod1, Pax3 and Runx1t1 in C2C12 cells seven days after transfection with 80 nM of indicated longRNA GapmeRs. (g) Relative expression of indicated genes in 3T3-L1 cells two days after transfection with LNAs against Bace1 versus scr across n = 4 independent experiments. If not stated otherwise, boxplots indicate median ± min/max and a paired two-tailed Student’s t-test was used to assess statistical significance. * = p < 0.05.