Fig 2: Validation of selected gene expression at protein level.a Expression maps presenting differential expression of 4 selected scRNA-seq DEGs (CXCL2, TBXT, CDH1, and CTNNB1) and ACTB (control gene) at single-cell resolution with a reference expression map. b qRT-PCR validation of fold-change values for the 4 selected DEGs at bulk-cell level; H460 (orange bars), Calu3 (red bars), and H1299 (green bars) compared with A549 (control cell line). X and y axes indicate gene names and fold-change values (log2), respectively. Fold-change values expressed as mean ± SEM from three separate experiments conducted in duplicate. Statistical significance; *P < 0.05, **P < 0.01, and ***P < 0.001. c Western blot analysis showing expression of proteins encoded by the 4 selected DEGs and Actin (loading control) in the four human NSCLC epithelial cell lines.

Fig 3: SOX2 is required for ß-catenin recruitment in neuromesodermal progenitors(A) Schematic of NMP differentiation in SOX2KD CRISPRi cells.(B) Immunostaining for SOX2, TBXT, and active ß-catenin on day 3 of NMP differentiation (scale bar represents 100 mm, n = 3).(C and D) Heatmap and metaplots of ß-catenin ChIP-seq signal in WT and SOX2KD NMP cells at ß-catenin peaks (n = 2) (C) that are lost upon SOX2 knockdown and (D) peaks that are unchanged. Table shows the percentage of peaks bound by ß-catenin, SOX2, or TCF (either TCF7L1, LEF1, or both).(E) Integration of RNA-seq (n = 3) and ChIP-seq datasets. *Significant overlap based on hypergeometric test, ChIP-seq peak overlap: p = 1.07 × 10-459; RNA-seq overlap: p = 5.64 × 10-352.(F and H) Metaplots showing nucleosome occupancy from ATAC-seq data at (F) ß-catenin peaks lost in SOX2KD or (H) ß-catenin peaks that are unchanged.(G and I) Violin plots quantifying ATAC-seq read densities. p values based on Wilcoxon rank-sum test. Dotted lines in (G) and (I) represent tag count threshold for significant ATAC-seq peaks.(J) Genome browser view of representative ß-catenin peaks of each category.(K) Model of SOX2 and WNT/ß-catenin interactions during NMP specification. See also Figure S6 and Table S5.