Fig 2: 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 3: Schematic showing increased conjugated BAs induce pathological lymphangiogenesis through an ROS-p90RSK-YAP-VEGFR3-dependent mechanism. Conjugated BAs bind to TGR5 expressed by the LECs and activate several key members of the redox or oxidative stress pathway. The activation of the redox pathway in turn produces ROS and activates the p90RSK via its phosphorylation at S380. Activated pp90RSK induced the SUMOylation of Prox1, and SUMOylated Prox1 increased the transcription of VEGFR3, ultimately increasing lymphangiogenesis. Conjugated BA-induced ROS also activated the YAP, which in turn increased the lymphangiogenesis. BA also enhanced the cellular metabolism by increasing the mRNA expression of the metabolic genes, namely PFKP, HK2, CO I, and FASN, that play roles in enhancing lymphangiogenesis. Generated with BioRender.com (Toronto, ON, Canada).

Fig 4: Lymphatic vessels exist deep in the mouse brain. (A) Schematic representation of experimental design of iDISCO+ tissue clearing procedure, immunostaining, and whole-brain imaging by light-sheet microscopy. (B) Light-sheet microscope fluorescent images of LYVE1 immunostaining of cleared whole-mount brain from different views. (C) Examples of optical sections taken from of whole-mount mouse brain from coronal (Y), sagittal (X), horizontal (Z), and orthogonal planes. Insets: Selected areas within the optical sections magnified to show the fluorescent signal of LYVE1 staining. (D) Schematic representation of experimental design of CLARITY tissue clearing procedure, immunostaining, and confocal microscopy imaging of thick brain sections. (E) Confocal images of LYVE1 fluorescent in cleared 1-mm-thick coronal brain section from mouse brain. Selected areas within the section magnified to show the fluorescent signal of LYVE1 staining in different areas. (F) Coimmunostaining of LYVE1 and CD34 in 1-mm coronal section. (G) Coimmunostaining of LYVE1 and CD235a in 16-µm brain section. (H) Same as (F) but coimmunostaining of LYVE1 and CD3. White arrows indicate T cells detected within the deep brain lymphatic vessels. (I) Coimmunostaining of LYVE1 and podoplanin (PDPN) in 16-µm brain section. (J) Same as (I) but coimmunostaining of LYVE1 and VEGFR3. (K) Same as (I) but coimmunostaining of LYVE1 and PROX1. DAPI, 4',6-diamidino-2-phenylindole.

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.