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.

Fig 4: IGFBP3 modifies part of the TBXT regulome.(A) IGFBP3 protein levels quantified by ELISA of UM-Chor1-Cas9 cells transduced with sgNTC or sgTBXT. Mean ± SEM of three biological replicates. Unpaired t test. ***P ≤ 0.001. (B) IGFBP3 protein levels in lysates and culture medium of UM-Chor1-Cas9 cells transduced with sgNTC or sgTBXT, with or without PNGase F treatment. (C) Western blot of UM-Chor1-Cas9 cells transduced with sgNTC, sgTBXT, or two sgRNAs targeting YAP1. (D) Viability of UM-Chor1 and U-CH2 spheroids expressing doxycycline-inducible IGFBP3 or EV, treated with doxycycline (0.5 μg/ml) versus untreated controls after 28 days. Mean ± SEM of two biological replicates (five to six spheroids averaged per condition). Representative spheroid images shown on the right. (E) Viability (luminescence units × 107) after 14 days of UM-Chor1-Cas9 spheroids double-transduced with sgNTC/sgNTC, sgTBXT/sgNTC, sgIGFBP3/sgNTC, or sgIGFBP3/sgTBXT. Mean ± SEM of three biological replicates (four to six spheroids averaged per condition). One-way ANOVA with Dunnett’s test for multiple comparisons; ***P ≤ 0.001. (F) Schematic of DIA-MS analysis in UM-Chor1-Cas9 cells 14 days posttransduction with sgNTC or sgIGFBP3, followed by T-DARPin D4 or E3_5, resulting in four conditions: ① control, ② TBXT inhibition, ③ IGFBP3 knockout, and ④ combined TBXT inhibition and IGFBP3 knockout. Created in BioRender. S.F. (2025) BioRender.com/r29p687. (G) MSigDB Hallmark GSEA comparing sgNTC (orange) versus sgIGFBP3 (purple) UM-Chor1-Cas9 proteomes under TBXT inhibition. The x axis shows NESs for gene sets significantly enriched in at least one condition (FDR < 5%, permutation test with Benjamini-Hochberg correction). NES values for both conditions are shown; dashed lines indicate FDR > 5%. (H) Expression changes of IGFBP3-dependent genes induced by T-DARPin D4 in sgNTC (black) or sgIGFBP3 (red and blue) UM-Chor1-Cas9 cells. Vertical lines indicate differences in log2(fold change). Red indicates reduced, blue increased expression upon IGFBP3 knockout (GLM with Wald test, n = 3, FDR < 1%). Interferon response genes are indicated and data points are circled. (I) Western blot of UM-Chor1-Cas9 cells with (sgIGFBP3) or without (sgNTC) IGFBP3 knockout and expressing DARPins E3_5 or D4.

Fig 5: T-DARPins are specific for TBXT and bind TBXT variants.(A) IP of Flag-tagged DARPins from U-CH2 cells with an anti-Flag antibody 5 days after transduction, followed by Western blotting (right). The whole-cell lysate input is shown on the left. One representative image of three independent experiments. (B) Enrichment of proteins identified by AP-MS of anti-Flag IPs for DARPins A2, B1, D4, and E3_5 and of anti-TBXT antibody (ab) IPs 4 days after transduction of U-CH2 and UM-Chor1 cells. Sphere sizes represent empirical Bayes moderated t statistic (n = 3) as an indicator of statistical certainty, and bait types and cell lines are indicated by colors. (C) Simulation of molecular interactions between T-DARPin structures predicted with AlphaFold2 and the TBXT DBD (PDB: 7HI8). T-DARPin:TBXT DBD (7H18) complexes were overlaid with the TBXT DBD:DNA PDB structure 6F58. The DNA is displayed as blue and red helices. (D) Anti-Flag bead pulldowns of DARPin E3_5 and T-DARPin D4 cotransfected into HEK293T cells with EV or a vector expressing HA-tagged WT TBXT, TBXT-R16L, TBXT-G177D, TBXT-H171R, or TBXT lacking the DBD (ΔDBD).