Fig 2: PROX1 and NKX2-1 co-immunoprecipitate with ASCL1 and have overlapping binding profiles in the genomes of SCLC-A cell lines(A) Proteins identified by mass spectroscopy of ASCL1 immunoprecipitates from NCI-H2107 cells shown ranked by spectral index. ASCL1 and its E-protein cofactors TCF3, TCF4, and TCF12 are shown, as are NKX2-1 and PROX1. See Table S2 for a complete list of proteins identified.(B) Immunoblots showing PROX1 and NKX2-1 co-immunoprecipitate with ASCL1 from NCI-H2107, NCI-H889, and NCI-H345 cells. White lines indicate non-informative lanes and regions omitted from the originating blots. The SCLC-N cell lines NCI-H524 and NCI-H2171 do not express ASCL1 and were used as negative controls. Input is the nuclear fraction from NCI-H2107. Western blots of lysates showing the presence of each TF in the cell lines used are shown in Figure S1.(C) RNA-seq (log2 FPKM) showing expression of ASCL1, NKX2-1, and PROX1, in SCLC tumors (T) or cell lines (C) in the specified SCLC subtype or NSCLC. The line indicates the mean and the asterisks indicate p values for each sample compared with SCLC-A tumors. *p < 0.05, **p < 0.01, ****p < 0.0001.(D and E) (D) ChIP-seq heatmap and (E) Venn diagram for ASCL1, PROX1 and NKX2.1 peaks identified in at least 2 of 3 SCLC-A cell lines (NCI-H2107, NCI-H128, and NCI-H889) identifies unique and shared genomic binding sites. See Table S3 for peak coordinates and Table S4 for TF-binding associated genes.(F) Top de novo motifs identified by HOMER in ChIP-seq peak regions. The text indicates the motif enriched, the factor family predicted to bind the motif, the percent incidence of the motif in ChIP-seq peak regions, and the p value significance of the specified motif.See also Tables S2 and S3.

Fig 3: MiR-204 inhibition reverses TTF1 overexpression-mediated proliferation, apoptosis, cycle arrest, migration, invasion, and autophagy in SK-N-BE cellsSK-N-BE cells were co-transfected with TTF1-overexpression plasmids and a miR-204 inhibitor.(A) MiR-204, TrkA, and TrkB expression was confirmed using RT-qPCR.(B) TrkA and TrkB protein expression was identified using Western blot.(C) LC3B-I, LC3B-II, Beclin1, p62, p53, Bcl-2, and C-Caspase3 protein expression was detected by Western blot in SK-N-BE cells after co-transfection.(D) Migration was measured using wound healing assays.(E) Cell proliferation was assessed using colony formation assays in co-transfected SK-N-BE cells.(F) Cell apoptosis was monitored and quantified using flow cytometry.(G) Cell proliferation was monitored using EdU staining.(H) Cell cycle distribution was determined using flow cytometry.(I) Invasion was investigated using Transwell assays. Magnification, 20×. Data are represented as mean ± SD. **p < 0.01, ##p < 0.01.

Fig 4: Impaired thyroid development following CDC42 ablation. (A-D) Sections through the thyroid bud of control (A,C) and conditional knockout (cko; B,D) embryos at E9.5 and E10.5 (t, thyroid bud). (E,F) Immunofluorescent staining for NKX2-1 in the thyroid buds of control and conditional knockout embryos. Scale bar=100 µm.

Fig 5: Identification of SE-associated genes in SCLC subtypes(A) The workflow used to identify expressed SE-associated genes including H3K27ac ChIP-seq, HiChIP, and RNA-seq.(B) Visualization of the H3K27ac ChIP-seq signal in a representative SCLC cell line from each subtype showing those that meet the SE criteria. Some TF encoding genes associated with the SEs are highlighted for comparison. NKX2-1 and NFIB are in gray because in the particular cell line shown, they just missed the stringent criteria used.(C) Human genome tracks showing the H3K27ac signal and HiChIP data associated with representative SE-associated genes shared and distinct across the cell lines. The black bars under the tracks indicate a region identified as an SE.(D) Venn diagram showing the number of SE-associated genes that are unique to or shared between the 3 SCLC subtypes. Total number of SE-associated genes is indicated. For an SE to be included in the analysis, it had to be present in 6 of the 11 SCLC-A, 4 of the 6 SCLC-N, or 3 of the 5 SCLC-P samples. An expression cut off of 5 FPKM in at least one relevant cell line was used as an expression filter. Data from SCLC cell lines are color-coded; SCLC-A (red), SCLC-N (blue), and SCLC-P (green).See also Table S1.