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STEM_1423_sm_SuppFigure1.pdf2064KSupplementary Fig. 1. Histology of Lin28a KO mice. (A) Some Lin28a KO mice have a cardiac defect. H&E images of Lin28a +/− and −/− P0.5 newborn hearts are shown. IVS interventricular septum, LA left atrium, LV left ventricle, RA right atrium, RV right ventricle, VSD ventricular septal defect. (B) Representative images of different organs/tissues in Lin28a +/− vs. −/− mice. Scale bars 200 μm (A), 100 μm (B). Related to Fig. 1.
STEM_1423_sm_SuppFigure2.pdf898KSupplementary Fig. 2. 7% of Lin28a −/− mice can survive to adulthood on a mixed background. (A) Representative images of Lin28a +/− and −/− mice at different ages. (B) Representative images of Lin28a +/− and −/− mice after DEXA scan. Related to Fig.1.
STEM_1423_sm_SuppFigure3.tiff769KSupplementary Fig. 3. Characterization of Lin28b KO and Lin28a +/−;b −/− mice. (A-B) Lin28b KO embryos and placentae show normal growth. N=9. (A) Weights of E18.5 embryos and placentae in Lin28b +/− vs. −/− mice. (B) Ratio of placenta/embryo weights in Lin28b +/− vs. −/− mice. (C) Organ weights normalized to total body weight in adult Lin28b +/− vs. −/− males. N=3. Epididymal fat pads were used to measure fat weights. (D) Weights of embryos and placentae of Lin28a +/+;b −/− vs. Lin28a +/−;b −/− embryos at E13.5. N=5-9. (E) Postnatal growth curve of Lin28a +/+ vs. +/− males. N=9. * p<0.05. Error bars represent SEM. Related to Fig. 2.
STEM_1423_sm_SuppFigure4.pdf1437KSupplementary Fig. 4. PCR genotyping confirms deletion of Lin28a (A-C) and Lin28b (D-F) alleles in various tissues of Lin28a fl/−; UBC-CreER (+) (A-C) and Lin28b fl/−; UBC-CreER (+) (D-F) mice. Tamoxifen was injected at E15.5 (A and D), P7-9 (B and E), or 6 weeks (C and F). Related to Fig. 4.
STEM_1423_sm_SuppFigure5.tiff770KSupplementary Fig. 5. Fetal Lin28a/b, not neonatal or adult Lin28a/b, regulates postnatal growth. (A-C) Postnatal growth curves of Lin28a fl/−; UBC-CreER (+) vs. Lin28a fl/− mice. Body lengths after tamoxifen (TAM) injections at (A) E15.5, (B) P7-9 and (C) 6 weeks. N=3-12. (D-F) Postnatal growth curves of Lin28b fl/−; UBC-CreER (+) vs. Lin28b fl/− mice. Body lengths after TAM injections at (D) E15.5, (E) P7-9 and (F) 6 weeks. N=5-13. Arrows indicate ages at TAM injection. * p<0.05, ** p<0.01. Error bars represent SEM. Related to Fig. 4.
STEM_1423_sm_SuppFigure6.pdf865KSupplementary Fig. 6. No expression of Lin28a/b in fat. Representative Lin28a (A) and Lin28b (B) immunostaining of wild-type fat tissues. Scale bars 100 μm. Related to Fig. 5.
STEM_1423_sm_SuppFigure7.pdf1304KSupplementary Fig. 7. Overexpression of let-7 in skeletal muscle is sufficient to cause growth retardation. (A) Let-7 levels in adult Lin28a +/− and KO mice. N=4. (B) Let-7 levels in adult Lin28b +/− and KO mice. N=3. (C-D) Postnatal growth curves of LSL-iLet-7s; Myf5-Cre(+) females given DOX from conception. N=4-13. (C) Body weights. (D) Body lengths. (E) Postnatal growth curve of Lin28a fl/fl; Myf5-Cre mice. Body weights. N=4-13. (F) Representative images of skeletal muscles in Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) mice. * p<0.05, ** p<0.01. Error bars represent SEM. Related to Fig. 6.
STEM_1423_sm_SuppFigure8.pdf1221KSupplementary Fig. 8. Characterization of Lin28a KO and Lin28b KO muscles. (A-B) Representative type II myosin immunostaining of gastrocnemius in Lin28a +/− vs. Lin28a KO (A) and Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) (B) mice. (C-D) Mitochondrial DNA content analyzed by quantitative PCR in gastrocnemius (gast) and quadriceps femoris (quad) of Lin28a +/− vs. Lin28a KO (C) and Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) (D) mice. Cox2 DNA was used for mtDNA and normalized to Rsp18 for relative amount calculation. N=3-4. (E-F) Nrf-1, Nrf-2, Pgc-1a, Pgc-1b, Ppara, Tfam, Tfb1m, and Tfb2m mRNA analyzed by quantitative RT-PCR in gastrocnemius of Lin28a +/− vs. Lin28a KO (E) and Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) (F) mice. N=3-4. Error bars represent SEM. Scale bars 100 μm. Related to Fig. 6.
STEM_1423_sm_SuppFigure9.tiff625KSupplementary Fig. 9. Hepatocyte- and pancreatic beta cell-specific Lin28b KO mice. (A-B) Postnatal growth curves of Albumin-Cre; Lin28b fl/fl males. N=7-9. (A) Body weights. (B) Body lengths. (C-D) Postnatal growth curves of Ins2-Cre; Lin28b fl/fl males. N=8-10. (C) Body weights. (D) Body lengths. Error bars represent SEM. Related to Fig. 6.
STEM_1423_sm_SuppFigure10.tiff1771KSupplementary Fig. 10. Targeted creation of a conditional Lin28b allele. (A) Upper: Genomic map of the Lin28b locus shows exons, restriction sites. Middle: Lin28b conditional targeting construct. Exon 2 is flanked by loxP sites. PGK-Neo cassette is flanked by a frt site. Lower: Targeted allele following a homologous recombination. Hi=HindIII, Xb=XbaI. (B) Southern blot showing ESC clones with the floxed allele. * denotes a non-specific band. (C) Western blot confirms no Lin28b proteins in Lin28b −/− ESC lysate. (D) PCR genotyping showing wild type (wt), floxed and deleted (null) alleles. Related to Experimental Procedures.
STEM_1423_sm_SuppTable1.tiff567KSupplementary Table. Numbers of Lin28a +/+;b −/− vs. Lin28a +/−;b −/− vs. Lin28a −/−;b −/− embryos collected at different ages. Numbers in parentheses show non-viable embryos, as defined by degenerative tissues or arrested heart. P-values were calculated by Fisher's exact probability test. Related to Fig. 3E.

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