Fig 2: shRNA-mediated targeting of MED10 in bladder urothelial carcinoma cells significantly attenuate their cancer stemness phenotype. (A) Heatmap showing the correlation between the expression of MED10, and stemness markers NANOG, SOX2, POU5F1/OCT4, CD44, PROM1/CD133, KLF4, and LIN28A in patients with urothelial recurrence or local recurrence from the AFFY_HG_U133_PLUS_2, GSE31684 cohort. Columns with similar annotations are collapsed by taking mean inside each group. Rows are centered; unit variance scaling is applied to rows. Both rows and columns are clustered using correlation distance and average linkage. 24 rows, 4 columns. (B) Immunohistochemistry photomicrographs of the differential expression of MED10, MKI67, OCT4, and LIN28A in benign, T1, T2, T3/4, or M1 tissue samples from the TMU-SHH UC cohort. Scale bar 200 µm. (C) Photomicrographs and histograms showing the effect of shMED10 on the formation of primary or secondary tumorspheres from SW1738 and JMSU1 cells. (D) Representative immunofluorescence images showing the effect of knocking down MED10 on the nuclear translocation and co-localization of OCT4A and LIN28A. DAPI served as nuclear marker. Scale bar 50 µm. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: Lin28a is downstream of Ephemeron and regulates Nanog expression.(A) Rex1-GFP flow cytometry profiles (Left) and colony formation capacity (Right) 40 hr post PD/LIF withdrawal for wild type and Eprn KO cells transfected with indicated siRNAs. (B) Rex1-GFP flow cytometry profiles and colony formation capacity 40 hr post PD/LIF withdrawal for wild type and Eprn KO cells transfected with Lin28a expression vector. (C) Rex1-GFP flow cytometry profile and colony formation capacity 40 hr post PD/LIF withdrawal with Nanog and Lin28a single or dual knockdowns in wild type cells. Quantification of percentage of GFP high cells were shown in (A-C). Percentage clonogenicity in (A-C) is measured by the number of AP positive colonies divided by the total number of cells plated, with representative AP staining images shown. Mean ± SD, n = 3. (D) Lin28a and Nanog expression relative to ß-actin upon PD/LIF withdrawal in Lin28a knockdown and control cells. Mean ± SD, n = 3. *p<0.05, Student’s t-test. (E) Correlation of Nanog and Lin28a protein expression immunostaining in wild type and Eprn KO cells 24 hr post PD/LIF withdrawal. (F) Representative images of cells co-immunostained with Nanog and Lin28a and quantified in E.DOI: http://dx.doi.org/10.7554/eLife.23468.012

Fig 4: The function of Wnt in embryo implantation is mediated through lin28a/let-7. a The efficiency of lin28a siRNA knockdown in blastocysts by immunostaining. b The relative lin28a mRNA level in lin28a siRNA knockdown embryo. c The relative expression of let-7a in lin28a knockdown but Wnt activation blastocyst. d Embryo attachment rate on fibronectin when embryo Wnt was inhibited and/or let7a was knockdown. e Embryo attachment rate on fibronectin when embryo Wnt was inhibited by DKK and/or lin28a was overexpressed (lin28a-TG). Data are means ± s.d. (N = 3–5), bar = 20 µm, p < 0.05 “*”, p < 0.01 “**”

Fig 5: Let-7 family members disturb embryonic EMT. a Immunochemistry staining of E-cadherin, b N-cadherin, c Active-ß-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