Fig 2: Expression correlation relationship of menin/MAPs genes and OXPHOS/glycolytic genes. (A) Workflow of the in silico correlation analysis of gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. (B) Heatmaps of the expression correlation between MEN1/selected 4 MAP genes and OXPHOS genes (upper) or glycolytic genes (lower) in normal (N) and tumor (T) samples. The genes are arranged from the highest to the lowest according to gene expression correlation coefficients of MEN1–OXPHOS genes or MEN1–glycolytic genes in breast tumors. (C) and (D) Scatter plots and linear regression analyses of MEN1/selected MAPs expression and mean expression of OXPHOS genes (C) or glycolytic genes (D) in normal and tumor samples. (E) Violin plots (lower panel) shows the average expressions of the genes of OXPHOS complexes I-V and glycolysis in the samples of each of the corresponding 4 groups are shown as violin plots. Based on the median values (where =median is “high” and KMT2A, MEN12, WAPL, and GATA3) and MEN1, the TCGA breast tumor samples were divided into 4 groups—1: high-low, 2: high-high, 3: low-low, and 4: low-high (upper panel). Letters on top of the violin plot denote statistical significance, where two groups with different letters are significantly different (p < 0.05) and those with the same letter are not.

Fig 3: Extended monolayer culture in CDBLY2 increases SIX2+ progenitor contribution to nephrons and proximalisation.a Confocal immunofluorescence of D7 + 14 (standard protocol) and D13 + 14 (extended protocol) organoids derived from the SIX2Cre/Cre:GAPDHdual lineage tracing iPSC line. Images depict merged and separated channels showing lineage-traced SIX2+ cells (mCherry; red), distal tubules (ECAD; green), podocytes (NPHS1; grey) and proximal tubules (LTL; blue). Scale bars represent 100 µm. b Flow cytometry of SIX2Cre/Cre:GAPDHdual lineage tracing organoids derived from extended (13 day + CDBLY2) and standard (7 day + E6 media) differentiations depicting SIX2-derived mCherry + (red) cell contribution to the EPCAM + (nephron) population. Flow plots shown in top panels are representative of the replicates across multiple experiments. Percentage mCherry contributions from flow cytometry are depicted in bar graph (bottom panel), where error bars represent SEM from n = 4 biological replicates across 3 independent experiments. Statistical significance was determined using an unpaired t test. Asterisks represent two-tailed P values adjusted for multiple comparisons using the Holm–Sidak method, alpha = 0.05 (*P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001 [Experiment 1: P = 0.000924; Experiment 2: P = 0.004899; Experiment 3: P = 0.004845]). Source data are provided as a Source data file. c Confocal immunofluorescence of D13 + 14 organoids demonstrating (left panel) aligned nephron morphology with nephron segmentation makers (nephron epithelium [EPCAM; green], distal tubule/Loop of Henle [SLC12A1; red], proximal tubules [LTL; blue], and podocytes [NPHS1; grey]) and (right panel) the presence of few GATA3 + connecting segment/ureteric epithelium structures (red), co-stained for nephron epithelium (EPCAM; green), podocytes (NPHS1; grey), and proximal tubules (LTL; blue). Inset in right panel shows GATA3 and LTL alone. Scale bars in (i) and (ii) represent 200 µm and 100 µm, respectively. d Confocal immunofluorescence of a D7 + 14 (standard) organoid depicting homogenous distribution of podocytes (NPHS1; grey), proximal tubules (LTL; blue), and nephron epithelium (EPCAM; green), as well as the presence of extended segments of centralised GATA3 + (red [nuclear]) connecting segment/ureteric epithelium (also highlighted in insets). Scale bar represents 200 µm.

Fig 4: EBN induces phosphorylation of GATA3 and its nuclear translocation. (A) After 16-h serum starvation, cultured differentiated keratinocytes were pre-incubated with or without SB203580 (10 µM) for 2 h, followed by different time of applied IL-4 (a positive control, 100 ng/mL) or EBN digest (100 µg/mL). Total GATA3 (T-GATA3) and phosphorylated GATA3 (P-GATA3) (both at ~48 kDa) were evaluated by western blot assays. (B) The quantification of the increased fold of P-GATA3/T-GATA3 expression levels from (A) after different time of applied IL-4 and EBN digest, with or without SB203580 pre-treatment, are shown. Values are expressed in mean ± SEM, n = 3. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control. (C) After 16-h serum starvation, cultured differentiated keratinocytes were pre-incubated with or without SB203580 (10 µM) for 2 h, followed by 240-min treatment of IL-4 (100 ng/mL, a positive control), EBN extract (100 µg/mL) and EBN digest (100 µg/mL). Phosphorylated GATA3 protein localization in keratinocytes was determined by immunofluorescence assays. n = 3. Bar = 20 µm. (D) The quantification of the percentage of nuclear to cytosol level of P-GATA3 intensities under different treatments from (C) are shown. Values are expressed in mean ± SEM, n = 3. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control or as indicated.

Fig 5: Differentiation of TGFß-inhibited naïve hPSCs transcriptionally recapitulates early trophectoderm specification in human embryos.(a) Diffusion maps of naïve cells during the SB time-course experiment, separated by days of treatment (left) and Louvain clustering (right). (b) Overlay of the diffusion maps with the relative expression of pluripotency markers NANOG, and POU5F1, and trophoblast markers CDX2, HAND1, GATA3. (c) Heatmap of the expression values of genes reported in (b) separated by the Louvain clusters. Note the overlap in the expression of pluripotency and trophoblast markers in cells within cluster D. (d) Correlation plot between pseudobulk data from Louvain clusters A/D/E and EPI (Epiblast), ICM (Inner Cell Mass), and TE+CTB (Trophectoderm+Cytotrophoblast) from cultured human pre-gastrulation embryos (Xiang et al., 2020). (e) PCA plot overlapping 200 randomly selected cells from each of the Louvain clusters A/D/E (individual dots) and data from 3D-cultured human pre-gastrulation embryos (Xiang et al., 2020), based on EPI, ICM, and TE+CTB cells (contour lines). PC1 variance 2.15, PC2 variance 1.41. (f) Heatmaps visualising the expression of genes in EPI, ICM, and TE+CTB (Xiang et al., 2020) and cells in Louvain clusters A/D/E. Note that the genes are in the same order for both plots. (g) Diffusion maps of naïve cells during the SB time-course experiment showing the relative expression of CTB markers – VGLL1 and PGF. (h) We propose there is a continuum of TGFß/Activin/Nodal signalling that spans a developmental window of human pluripotent states from naïve to primed. In both states, active TGFß signalling promotes the expression of common pluripotency genes, such as NANOG and POU5F1, and contributes to the maintenance of pluripotency. SMAD2/3 are additionally required in naïve hPSCs to sustain the expression of naïve pluripotency factors, including KLF4 and DNMT3L. Inactivating TGFß signalling in naïve hPSCs leads to the downregulation of pluripotency genes, thereby enabling the induction of trophoblast differentiation.