Figure 4. Lin28 is a predicted target of miR-125b in differentiating hESCs.

A) In silico analysis of potential miR-125b targets using Target Scan Human identified Lin28 as a potential target. Seed match refers to the match between a sequence in the 3′ untranslated region (UTR) of the putative target gene and positions 2–8 of the mature miRNA; site-type contribution reflects the average contribution of each site identified; 3′ pairing contribution describes the impact of miRNA-target complementarity outside the seed region; local AU contribution is a measure of the target transcript AU content 30 nucleotides upstream and downstream of the predicted site; position contribution reflects the distance to the nearest end of the target's UTR. For each of these criteria, a more negative score indicates a more favorable site. The context score is the sum of the contributions of site-type, 3′ pairing, local AU, and position criteria. Conserved branch length is the sum of phylogenetic branch lengths between species that contain the site. The probability of conserved targeting (P_CT) reflects the Bayesian estimate of the probability that a site is evolutionarily conserved through selective advantage, rather than by chance. These criteria applied to miR-125b/Lin28 were similar to those for the established let-7a/Lin28 interaction. B) Conservation among mammals of the miR-125b binding site in the 3′ UTR of Lin28. Bta, cow; Cfa, dog; Cpo, guinea pig; Dno, armadillo; Eca, horse; Eeu, hedgehog; Gga, chicken; Hsa, human; Laf, elephant; Mdo, opossum; Mml, rhesus; Mmu, mouse; Ocu, rabbit; Ptr, chimpanzee; Rno, rat; Sar, shrew. C) Undifferentiated αMHC-GFP reporter hESCs (Undiff) and αMHC-GFP⁺ CMs sorted from reporter hESCs grown in differentiation medium for 8 or 14 days were evaluated for Lin28 expression by qPCR. Endogenous expression of Lin28 mRNA declined with CM differentiation. Data shown are mean±s.e.m. (N = 3). ***, p<0.001.