Fig 2: Lin28-derived tumors harbor proliferating CM cells. (A) Overexpression of CM-specific transcription factors in the tumor. Microarray data (tumor, n = 4; control, n = 4). (B) Six2 and Ki67 immunohistochemistry in normal kidneys and LIN28a-derived tumors. Bar, 100 µm. (C) Histology of the E18.5 nephrogenic zone and adult tumors. Note that embryonic structures consisting of a branched UB surrounded by CM cells appears in the adult tumors. Bar, 50 µm. (D) Six2 and Keratin8 coimmunostaining in a normal embryonic kidney, a normal adult kidney, and an adult tumor. (Note: Keratin 8 is a marker for UB cells during kidney development and for the collecting duct in the adult kidney). In contrast to normal adult kidneys, in which Six2 is not expressed and keratin8 expression is restricted to the collecting duct, the expression pattern of Six2 and Keratin8 in the tumors is similar to the nephrogenic zone during kidney development. Bar, 100 µm.

Fig 3: MeCP2 knockdown re-activates LIN28A expressionLIN28A mRNA and protein levels were detected by Real-time PCR or western blotting in PANC1 A. and PaTu8988 B. cells transfected with sh-MeCP2 or sh-EGFP plasmids, as well as MBD3 knockdown in PANC1 C. and PaTu8988 cells D. Data are presented as mean±SD. *, P < 0.05.

Fig 4: The LIN28/let-7 pathway cooperates with Ctnnb1 mutation to drive invasive intestinal adenocarcinoma. (A) Representative image of the small intestines in wild-type (WT), iLin28a, and iLIN28B animals 6–7 mo after doxycycline. Intestinal tumors are indicated by asterisks. (B) Representative H&E images of the iLin28a and iLIN28B intestinal adenocarcinoma with flat morphology (top panel) and invasive features (bottom panel). Arrowheads indicate the invasive ends growing into the muscularis. Bars: top panel, 200 µm; bottom panel, 100 µm. (C) Representative immunohistochemistry staining against phostpho-Stat3 (Y705) of normal mucosa and iLin28a and iLIN28B intestinal tumors. Bar, 100 µm. (D,E) Quantitative PCR (qPCR) analysis showing up-regulation of tumor progression genes in iLin28a and iLIN28B tumors compared with wild-type and adjacent mucosa. Data are mean ± SD; n = 3–5 from each genotype. (F,G) qRT–PCR analysis measuring the levels of mature let-7 miRNAs in wild-type intestinal mucosa, iLIN28 adjacent mucosa, and iLIN28 intestinal tumors. Data are mean ± SD; n = 3 from each sample type. (H,I) qPCR analysis showing up-regulation of Wnt target genes in iLin28a and iLIN28B tumors. Data are mean ± SD; n = 3–5 from each sample type. (J) Representative immunohistochemistry staining against Ctnnb1 (ß-catenin), Cd44, and Sox9 of normal mucosa and iLin28a and iLIN28B intestinal tumors. Bar, 100 µm. (K) Tumor multiplicity of iLIN28B and iLIN28B; iLet-7 animals 7 mo after doxycycline. Data are mean ± SD; n = 3 from each genotype, and the animals are littermates. (L) Percentage of the tumors with mutations detected in the indicated genes. (M) Summary of the mutant heterozygous allele frequency and mutation position in all of the tumor samples with Ctnnb1 somatic mutation.

Fig 5: Correlation graphs and ROC curves for candidate biomarkers of neural dysfunction in IEM patients. (a) Graphs showing changes in BDNF and threonine in plasma of patients with deficits in psychomotor development and the ROC curves for different biomarker combinations calculated comparing affected with non-affected IEM patients. (b) ROC curves for THBS3 mRNA in blood and AABA in plasma and its combination for the detection of deficits in attention and/or executive functions calculated comparing affected with non-affected IEM patients. (c) Graphs showing changes in LIN28A mRNA in blood and alanine and cysteine in plasma of patients with other behavioral symptoms and the ROC curves for LIN28A. THBS3 and alanine biomarker combinations calculated comparing affected with non-affected IEM patients.