Fig 2: VEGFA and BMP2 adenoviral vectors (ad) respectively upregulated the expression of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein 2 (BMP2) in BMSC. Representative immunofluorescence images of BMSC in monolayer transduced with ad-GFP (a), ad-VEGFA (b), and ad-VEGFA + BMP2 (c–e) adenoviral particles. BMSC (5× 104) were seeded in each scaffold and harvested after 3 and 14 days for mRNA and protein analyses. Significant upregulation of VEGFA mRNA (f) and VEGFA protein (h) levels were found at both time points in both ad-VEGFA and ad-BMP2 + ad-VEGFA BMSC compared with the ad-GFP BMSC. Similarly, significant upregulation of BMP2 mRNA (g) was found in ad-BMP2 + VEGFA BMSC compared with the ad-GFP and ad-VEGFA BMSC at both time points. VEGFA and BMP2 mRNA levels were normalized to GAPDH mRNA level. i ELISA disclosed higher levels of secreted BMP2 in the culture supernatant of ad-BMP2 BMSC compared with the ad-GFP BMSC at both 3 and 14 days. Error bars in (f) and (g) represent SEM of three repeated experiments (n = 3) performed in three technical replicates. ANOVA test with Bonferroni post hoc analysis was used for statistical analysis in (f) and (g) and Student’s t test was performed in (i). Error bars in (i) represent SEM of three repeated experiments (n = 3). ***p < 0.001, **p < 0.01. GFP green fluorescent protein, ns not significant

Fig 3: Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) co-expression-mediated bone formation is associated with upregulation of osteogenic and angiogenic markers in the in vivo scaffold explants. Custom PCR array was used to examine differentially expressed osteogenesis- and angiogenesis-related genes in scaffold explants at 2 weeks. ANOVA revealed upregulation of a number of osteogenesis- and angiogenesis-related genes such as a ALPL, b RUNX2, c SPP1 (osteopontin), d ANGPT1, and e PGF in ad-BMP2 + VEGFA explants compared with the ad-GFP and ad-VEGFA explants. ANOVA test with Bonferroni post hoc analysis was undertaken for statistical analysis. **p < 0.01, **p < 0.05. GFP green fluorescent protein, ns not significant

Fig 4: VEGFA analysis. (A) Western Blot analysis of VEGFA on eye cup samples of all the experimental groups. Statistical analysis was performed by one-way ANOVA test followed by Tukey test. Data are shown as mean ± SE (n = 4). *p < 0,05; **p < 0,01; ***p < 0,001 versus Control. #p < 0,05; ##p < 0,01; ###p < 0,001 versus LD. Original western blot presented is available in Supplementary Fig. S1. (B) Confocal images of retinal cryosections immunolabelled for VEGFA (red). Nuclei were stained with Bisbenzimide (blue). The images refer to the central dorsal area of the retina. VEGFA increased in the rat retina after light exposure over time. CTRL (Control); LD (Light damage); LD + 7rec (Light damage + 7 days of recovery); LD + 60rec (Light damage + 60 days of recovery); LD + 120rec (Light damage + 120 days of recovery); ONL (Outer nuclear layer); INL (Inner nuclear layer); GCL (Ganglion cell layer).

Fig 5: The interaction between GSE1 and TACSTD2 drives metastatic disease, castration resistance, and disease progression in patients with prostate cancer. Box-and-dot plots showing the differential expression of (A) GSE1 and (B) TACSTD2 genes in normal, tumor, and metastatic samples from the TCGA PRAD cohort. Box-and-whisker plots showing the correlation between (C) GSE1 or (D) TACSTD2 gene expression and clinical M stage in the TCGA PRAD dataset. (E) Graphical representation of the correlation between GSE1 and TACSTD2 gene expression levels. (F) Principal component analysis of the expression data for primary localized PCa vs. castration-resistant bone metastatic prostate cancer in the GSE32265 cohort. Unit-variance scaling was applied to rows; SVD with imputation was used to calculate principal components. X- and Y-axes show Principal Components 1 and 2, which explain 26.6 and 13.2% of the total variance, respectively. Prediction ellipses were computed such that, with a probability of 0.95, a new observation from the same group would fall inside the ellipse. N = 55 data points. (G) Expression heatmap showing the relationship between metastasis, the TMPRSS2-ERG fusion gene, and expression of GSE1, TACSTD2, KLK3/PSA, and biomarkers of angiogenesis, metastasis, and proliferation in the GSE32265 cohort. Columns with similar annotations were collapsed by taking the mean in each group. Rows are centered; unit-variance scaling was applied to rows. Both rows and columns were clustered using correlation distance and average linkage, with 25 rows and 5 columns. (H) Representative Western blot images of the differential expression of GSE1, TACSTD2, VIM, SNAI2, and VEGFA proteins in HPrEC, LNCaP, PC3, or DU145 cells. GAPDH served as a loading control. FC, fold change; meta, metastatic; norm, normal; M, distant metastasis; M0, no distant metastasis; M1a, distant metastasis to non-regional lymph node(s); M1b, distant metastasis to bone(s); M1c, distant metastasis to other site(s) with or without bone disease; BCR, biochemical recurrence; SVD, singular value decomposition. ** p < 0.01; *** p < 0.001; ns, not significant.