Fig 2: hiPSC-ECs Carrying the SERPINE1-FOSB Translocation Show Increased FOSB Expression(A) Schematic overview of the differentiation protocol and purification of ECs from hiPSCs.(B) Bright-field images showing typical EC morphology of hiPSC-ECs. Scale bar represents 500 µm.(C) Fluorescence-activated cell sorting (FACS) analysis of EC marker expression on isolated ECs at passage 3 (P3) from hiPSC-ECsWT (black-filled histogram) and hiPSC-ECsSERPINE1-FOSB (D3) (red-filled histogram), and relevant isotype control (gray-filled histogram).(D) Quantification of normalized relative surface expression levels (MFI) of VEC, VEGFR2, VEGFR3, CD31, CD34, and CD105. n = 3 (biological replicates, 3 independent batches of hiPSC-ECs). Error bars are SDs, *p < 0.005.(E) Real-time qPCR analysis of FOSB expression in hiPSCsWT, hiPSCsSERPINE1-FOSB (D3), hiPSC-ECsWT, and hiPSC-ECsSERPINE1-FOSB (D3) normalized to the housekeeping gene HPRT1 (×1,000). n = 3 (biological replicates, 3 independent batches of hiPSC-ECs). Error bars represent means ± SDs.(F) Western blot of FOSB expression in hiPSC-ECsWT and hiPSC-ECsSERPINE1-FOSB (D3). Short and long exposure of the gel is shown. USP7 was used as a housekeeping control.

Fig 3: Functional Assessment of hiPSC-ECs Carrying the SERPINE1-FOSB Translocation(A) Analysis of hiPSC-ECsWT and hiPSC-ECsSERPINE1-FOSB (D3) proliferation rates when cultured in basal endothelial cell growth medium supplemented with 1% PPS (1%), 1% PPS supplemented with 50 ng/mL VEGF (1% VE), or complete EC growth medium (full) for 24 h. Proliferation was determined by using a Presto Blue assay. n = 3 (biological replicates, 3 independent batches of hiPSC-ECs). Error bars are shown as SDs; ∗p < 0.0001 and ∗∗p < 0.0005.(B) Representative images of Matrigel tube formation assay using hiPSC-ECsWT and hiPSC-ECsSERPINE1-FOSB (D3) at the 48-h time point. Scale bar represents 500 μm. The right panel shows the quantification of the number of junctions and meshes. Error bars are SDs; ∗p < 0.005 and ∗∗p < 0.001.(C) Representative absolute resistance of the EC monolayer in complete EC growth medium. N = 6 (2 independent experiments with 3 batches of hiPSC-ECs). Errors bars are shown as means ± SDs.(D) Normalized resistance (4 kHz) of the EC monolayer in complete EC growth medium. N = 6 (2 independent experiments with 3 batches of hiPSC-ECs). Error bars are shown as means ± SDs; ∗∗∗p < 0.001.(E) Representative immunofluorescent images of CD31 and ZO1 to analyze the cell tight junctions. Merged images show CD31 in red, ZO1 in green, and DAPI in blue. The right panels show further enlarged areas selected from the shown images (dashed squares). Scale bar represents 50 μm.

Fig 4: BT2, T4, and T6 block endothelial FosB/?FosB expression, proliferation, migration and wound repair after in vitro injury and network formation.(A) HMEC-1 cells were serum-arrested for 20 hours, treated with 30 µM compound in serum-free medium (SFM) (without EGF/hydrocortisone) for 4 hours, and stimulated with 10% fetal bovine serum (FBS) (with EGF/hydrocortisone) and compound (same concentration) for 1 hour. Western blotting was then performed with independent biological duplicates where indicated. Approximate positions of molecular weight markers are shown. Data represent three biologically independent experiments. Veh, vehicle. (B) Serum-deprived HMEC-1 cells were treated with compound in 5% FBS (with EGF/hydrocortisone), and proliferation was monitored using xCELLigence. Top: Representative real-time profiles from one experiment. Cell index is a quantitative measure of cell growth. Bottom: Data represent means ± SEM of five to eight independent experiments after 79 hours. One-way analysis of variance (ANOVA). (C) BAECs in 10% FBS were seeded into wells containing 0.8-µm Transwell inserts. After 48 hours, medium was changed to 0.01% FBS for 48 hours. Compounds (1 µM) were added to the upper chamber in 0.01% FBS, and medium in the lower chamber was changed to 10% FBS and VEGF-A165 (50 ng/ml). Cells were left for 24 hours. Typical 4',6-diamidino-2-phenylindole–stained nuclei are shown. Nuclei were quantified with National Institutes of Health (NIH) ImageJ. Data represent means ± SEM of four to five independent experiments. Kruskal-Wallis multiple comparisons test. (D) Scraped HMEC-1 monolayers were treated with 0.6 µM compound in 5% FBS. Regrowth in denuded area was measured 48 hours after scraping. Representative regrowth from one experiment is shown. Regrowth area was determined with Image-Pro Plus. Data represent means ± SEM of five independent experiments. One-way ANOVA. (E) HMEC-1 cells in 1% FBS and fibroblast growth factor 2 (FGF-2; 50 ng/ml) were mixed with compound (3 µM) and seeded in wells coated with Matrigel. Network formation over 24 hours was quantified using Image-Pro Plus. Data represent means ± SEM of five to six independent experiments. Kruskal-Wallis multiple comparisons test. Representative network formation on Matrigel from same control well over time is shown. Where indicated, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Expression of FOSB in human glioma samples and glioma cells. (A) FOSB expression in normal brain samples, low-grade glioma, and high-grade glioma samples was determined by Western blot (n = 5 each). (B) FOSB expression in normal human astrocytes and five glioma cell lines (U251, U118, U87, T98G, and A172) was compared in vitro (n = 3). (C) The quantitative analysis of (A) showed high FOSB expression in high-grade gliomas (n = 5, **P < 0.01 vs. normal brain tissues; ##P < 0.01 vs. WHO II glioma tissues). (D) The quantitative analysis of (B) showed higher expression of FOSB in glioma cell lines (n = 3, **P < 0.01). (E) FOSB expression in cell lines was detected by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). FOSB expression in glioma cell lines (U251/U118/U87/T98G/A172) was also higher than that in human astrocytes (HA; n = 3, **P < 0.01). (F) Immunohistochemistry was used to detect expression in low-grade and high-grade glioma samples (n = 3). (G) The positive cell count of (F) showed high FOSB expression in high-grade gliomas (n = 3, *P < 0.01 vs. low-grade glioma). (H) Hematoxylin-eosin staining of low-grade and high-grade glioma (n = 5).