Fig 2: DATS promotes lymphatic endothelial cell proliferation, tube‐like structure formation, and Akt phosphorylation. A, Free H2S levels in LECs treated with/without DATS. Data are mean±SEM (n=6 each). *P<0.05 vs control by Student t test. B, WST‐1 proliferation assay of LECs in response to DATS (n=7 each). ****P<0.0001 vs 0 μmol/L, #### P<0.0001 vs 10 μmol/L by 1‐way ANOVA and Tukey post hoc tests. C, Representative images of DATS‐induced tube formation in LECs. D, Quantitative analysis of total tube lengths. Data are mean±SEM (n=10 each). ***P<0.001 vs control by Student t test. E, Representative immunoblots of p‐Akt, Akt, PROX1, β‐Actin, and GAPDH after DATS treatment. F, Quantitative analysis of Akt phosphorylation. Data are mean±SEM (n=7 each). **P<0.01 vs 0 μmol/L and # P<0.05 vs 10 μmol/L by 1‐way ANOVA and Tukey post hoc tests. G, Quantitative analysis of PROX1. Data are mean±SEM (n=5 each). **P<0.01 vs 0 μmol/L and ## P<0.01 vs 10 μmol/L by 1‐way ANOVA and Tukey post hoc tests. H, Level of VEGF‐C concentration in LECs after DATS treatment detected by an ELISA kit. Data are mean±SEM (n=6 for each). I, Representative immunoblots of p‐Akt, Akt, PROX1, and GAPDH at sequential time points after DATS treatment. J, Quantitative analysis of Akt phosphorylation. Data are mean±SEM (n=5 each). **P<0.01 and *** P<0.001 vs 0 minutes by 1‐way ANOVA and Tukey post hoc tests. K, Quantitative analysis of PROX1. Data are mean±SEM (n=5 each). *P<0.05 and *** P<0.001 vs 0 minutes by 1‐way ANOVA and Tukey post hoc tests. Akt indicates protein kinase B; DATS, diallyl trisulfide; H2S, hydrogen sulfide; LECs, lymphatic endothelial cells; N.S., not significant; p‐Akt, phospho‐Akt; PROX1, prospero homeobox protein‐1; t‐Akt, total Akt; VEGF‐C, vascular endothelial growth factor‐C; and WST‐1, water soluble tetrazolium salts‐1.

Fig 3: Conjugated BAs promote ROS production and induces p90RSK-mediated Prox1 SUMOylation and enhances VEGFR3. TCA (100 µM) induced (A) cellular ROS production at 45 min and (B) mtROS production after 12 h of treatment in HLEC, measured by fluorometric assay in plate reader. TGR5 antagonist SBI-115 (10 µM) pre-treatment for 1 h inhibited the TCA-induced mtROS production. (C) Western blot analysis showed that TCA (100 µM)-induced p90RSK phosphorylation in HLECs after 24 h, which was inhibited by p90RSK inhibitor BI-D1870. (D) After 24 h treatment with TCA (100 µM), the Prox1 was immunoprecipitated using anti-Prox1 antibody and probed with anti-SUMO1 antibody. TCA treatment induced Prox1 SUMOylation in HLECs which was inhibited by inhibition of p90RSK activation by using BI-D1870. (E) TCA (100 µM) induced VEGFR3 mRNA expression which was inhibited by p90RSK inhibitor BI-D1870 and ROS inhibitor NAC. (F) Prox1 gene was knocked down in HLECs by siProx1 and (G) Prox1 knockdown inhibited the TCA-induced VEGFR3 expression. (H) TCA induced tube formation of HLECs in Matrigel at 4 h, which was inhibited by the TGR5 antagonist SBI-115, p90RSK inhibitor BI-D1870. (I) TCA (100 µM) induced the HLEC invasion in 3D collagen matrix at 20 h and was inhibited by BI-D1870 or TGR5 antagonist SBI-115. Values represent mean ± SD, n ≥ 3. Statistical analysis was done by ANOVA followed by Fishers LSD. *, **, ***, **** indicates p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, p ≤ 0.0001, respectively, as compared to respective control., $$$, $$$$ indicates p ≤ 0.001, p ≤ 0.0001, respectively, as compared to TCA. #, ###, #### indicates p ≤ 0.05, p ≤ 0.001, p ≤ 0.0001, respectively, as compared to TCDCA.

Fig 4: Gene associations and regulation of FOXA2 expression.a FOXA2 IHC staining of primary SCLC. Representative images of a FOXA2-negative region and a FOXA2-positive region are shown. Scale bar: 500 μm and 100 μm (inset). b scRNA-seq UMAP including 19 clinical metastatic SCLC tumors indicating FOXA2+ and FOXA2- cells. c Violin plot of FOXA2 expression by scRNA-seq in each of these 19 cases. d Pulmonary neuroendocrine cell signature expression in FOXA2+ cells and FOXA2- cells assessed by scRNA-seq (** < 0.01, P = 0.0000, one-sided Mann–Whitney U test). e Ranking plot of genes highly expressed in FOXA2+ vs. FOXA2- cells at the single-cell level. f Correlation plot of nominated top 100 genes from panel (e) assessed relative to FOXA2 expression in bulk RNA-seq data. g Heatmap of co-expression on ASCL1, FOXA2, and PROX1 in scRNA-seq. h ASCL1 KD effect on protein expression of PROX1, FOXA2, and GAPDH (control) in H1836 and SHP-77 cell lines. The samples derive from the same experiment but different gels for ASCL1, FOXA2, another for PROX1 and GAPDH were processed in parallel. Western blot result is a representative data of one of the 3 individual experiments. i, j ASCL1 ChIP-seq at the FOXA2 and PROX1 gene loci in 2 SCLC cell lines (H1836 and SHP-77). Two independent replicates using anti-ASCL1, and two isotype (IgG) controls are shown. Highlighted regions indicate promoter and enhancer domains. k PROX1 KD and protein expression of FOXA2 and GAPDH (control) in H1836 and SHP-77 cell lines. The samples derive from the same experiment but different gels for FOXA2, another for PROX1 and GAPDH were processed in parallel. Western blot result is a representative data of one of the 3 individual experiments.

Fig 5: ASCL1 binds to the FOXA2 locus in met-associated primary SCLC.a Immunofluorescence (IF) image of never-met primary and met-associated primary tumor clinical samples stained for DAPI, ASCL1, PROX1, and FOXA2 for 2 cases. Insets are indicated in white box. Scale bar: 1000 μm (overlay), 100 μm (DAPI, ASCL1, FOXA2). b Schematic of FFPE-ATAC-seq from never-met primary and met-associated primary tumor clinical samples. Created in BioRender. Rudin C. (2025) https://BioRender.com/mlreujw. c Chromatin accessibility assessed by ATAC-seq at the FOXA2 locus (Peaknorm). Highlighted regions indicate promoter and enhancer domains. d Schematic of ATAC-seq and ChIP-qPCR on PDX tumors. Created in BioRender. Rudin C. (2025) https://BioRender.com/mlreujw. e ATAC-seq comparing ASCL1+FOXA2+ PDX (Lx101, Lx328, Lx891) vs ASCL1+FOXA2- (Lx1231) PDX tumors at the FOXA2 locus. f IgG and ASCL1 ChIP-qPCR of the FOXA2 locus, and negative control region in one ASCL1+FOXA2- (A + F-) and three ASCL1+FOXA2+ (A + F + ) SCLC PDX models from panel. Bars are shown in Mean±S.D of N = 3 experimental replicates. g Schematic of ASCL1 interaction with FOXA2 in never-met versus met-associated SCLC. Created in BioRender. Rudin C. (2025) https://BioRender.com/j34m841.