Fig 2: (A) Lin28A expression profile in pediatric CNS cancer cell lines. SDS-PAGE on 10% polyacrylamide gel of total cell lysates from untreated cells harvested at 80%–90% confluency. Samples loaded with volumes for 30 μg protein. Lin28A protein was detected using the anti-Lin28A antibody (#8706; Cell Signaling Technology) at 1:2000 dilution. T47D: adult breast cancer (positive control for Lin28A expression); BT-183: embryonal tumors with multilayered rosettes (ETMR) (positive control for Lin28A expression); IMR5 and LAN1: pediatric neuroblastoma; KCCF1, BT12, and BT16: pediatric atypical-teratoid rhabdoid tumor (AT/RT) (negative controls for Lin28A expression); YP-MEL: malignant melanoma derived from neurocutaneous melanosis (NCM); NCM patient cells: derived from a 2-year old male with multiple, large congenital nevi (B) IC50 concentrations of compound c1632 in panel of CNS cancer cell lines. Lin28A expression in the presence of c1632 correlates with the sensitivity of cells to Lin28-specific inhibitor c1632. Cells were cultured in the presence of the c1632 at increasing concentrations and cell viability was measured after 96 h. All the data shown are representative of three replicates. (C) Effect of compound c1632 (#6068; Tocris Bioscience) on Lin28A protein levels, 96-h post-treatment. Lin28A positive cell lines (T47D, BT-183 and YP-MEL) demonstrated successive reduction in Lin28A protein in the presence of c1632. Lin28A expression levels in the presence of c1632 in each cell line were calculated by normalizing β-Actin protein loading controls. (D) Pharmacological inhibition of Lin28A using c1632 and siRNA targeting Lin28A (OnTarget Plus SMARTpool L-028584-01; Thermo Scientific)7 transiently transfected into cells using Lipofectamine 2000 (Invitrogen), both leads to increase in the expression of let-7a miRNA (Taqman MicroRNA Assay: has-let-7a: 000377)3 in NCM (YP-MEL and patient cells) and ETMR (BT-183) cells compared to treatment with non-targeted vector control siRNA, measured by Taqman miRNA qRT-PCR. Change in miRNA expression levels were relative to noncoding RNU6B7 (E, F) Knockdown of Lin28A leads to significant reduction of oncogenic hallmarks such as tumor initiation and progression (PI3K signaling) [#4228, Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody; #4257, PI3 Kinase p85 (19H8) Rabbit mAb, Cell Signaling Technology], tumor survival (IGF-1R signaling) [#4568, Phospho-IGF-I Receptor β (Tyr980) (C14A11); #3018, IGF-I Receptor β (111A9), Cell Signaling Technology] and tumorigenic evasion of immune surveillance (PD-L1 expression) [#13684, PD-L1 (E1L3N) XP Rabbit mAb, Cell Signaling Technology) in NCM and ETMR tumor cells.

Fig 3: Let-7 family members disturb embryonic EMT. a Immunochemistry staining of E-cadherin, b N-cadherin, c Active-β-catenin, d Lin28a in control let-7 g and let-7 g overexpressed embryo (Let-7 g-TG) after embryo transfer. EPI: epiblast. TE: trophectoderm. EXE: extra-embryonic ectoderm. Scale bar: 60 μm

Fig 4: Characteristics of LIN28B binding on non-miRNAs(A) Changes in read density at any binding site can be plotted as a function of LIN28B expression level, yielding a regression and slope. The slope is correlated to strength of LIN28B binding.(B) Distribution of slopes (μ), encompassing 11,433 target sites.(C) Frequency of known LIN28 motifs, ‘‘UGAU,’’ in eCLIP-identified binding sites.(D) Frequency of known LIN28 motifs, ‘‘GNNG,’’ in CLIP-identified LIN28 binding sites.(E) Boxplots showing the distribution of GC, GU, AU, and GG pairs found in RNA structures formed in LIN28 binding sites.(F) Cumulative distribution functions showing the minimum free energy of the ensemble for all higher-affinity binding sites (m < 0, solid red), the 100 highest-affinity binding sites (dashed red), all lower-affinity sites (m > 0, solid green), and the 100 lowest affinity binding sites (dashed green) compared with randomly chosen sequences outside of eCLIP-identified binding sites (gray).(G) Receiver-operator characteristic for two random forest classifiers that were developed to predict the affinity of a LIN28 binding site (blue) or the location of a LIN28 binding site (yellow). Predictions against data with shuffled identifiers were used as a control (dashed).(H) Boxplots showing the accuracy of random forest classifier predictions compared with performance on a dataset with shuffled identifiers.

Fig 5: C1632 protects against high-fat diet (HFD)-induced NAFLD in mice.a, b Representative immunoblot and quantitative densitometric analyses for Lin28A, Lin28B, NCoR1 and SREBP-1 (precursor and mature form) in livers from 17-week-old male wild-type mice fed with HFD treated with vehicle or C1632 (50 mg/kg) after 4 weeks of treatment (5 IP injections per week). α-Tubulin was used as a loading control (n = 3 mice/group, Lin28A, *P = 0.0272; Lin28B, *P = 0.0258; NCoR1, *P = 0.0167; M-SREBP1, *P = 0.0467). c Representative Hematoxylin and eosin (H&E) staining and Perilipin (PLIN2) staining of liver sections from vehicle and C1632 (50 mg/kg)-treated mice fed with a HFD. The arrowheads indicate ballooned hepatocytes. Scale bars correspond to 500 μm. d–f Lipid droplet size and number were quantified by Image J (n = 8 histological images/group, d, ***P = 0.0004; e, **P = 0.0018; f, **P = 0.0034). g Hepatic triglyceride (TG) content in mice fed a HFD and treated with vehicle or C1632, as described in (a) (n = 7 mice/group; *P = 0.0175). h–k Serum TG (n = 8 mice/group), FFA (n = 7 mice/group), insulin (n = 7 mice/group), and β-OHB levels (n = 4 mice/group, *P = 0.0407) in mice fed with a HFD and treated with vehicle or C1632, as described in (a). l–n Relative mRNA levels of lipogenesis-, ketogenesis-, inflammation- and fibrosis-related genes in livers from mice fed with a HFD and treated with vehicle or C1632 as described in (a) (n = 7 mice/group, Acl, **P = 0.0011; Acc, **P = 0.0015; Pparα, *P = 0.0184; Cpt1a, **P = 0.0087; Hmgcs2, *P = 0.0489; Hmgcl, *P = 0.0410; Tnfα, *P = 0.0160; Ccl2, *P = 0.0308; Cxcl10, *P = 0.0489; Col1a, **P = 0.0097). β-Actin was used as a housekeeping gene. o Serum ALT levels in mice fed with a HFD and treated with vehicle or C1632 as described in (a) (n = 5 mice/group, *P = 0.0227). Numbers to the left of the blots indicate molecular weight markers. Values are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Groups were compared using two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.