Fig. 7: Proteomic analysis of the BVO secretome.

a Pathway enrichment and (b) volcano plot of proteins detected in the secretome of BVOs treated with DMSO (CTR) or PFK15 (2.5 µM) for 24 h. n = 5 BVOs per pooled sample, from 2 separate preparations. P-values < 0.05 shown in red. Volcano plot was generated using GraphPad Prism 9 software and statistical comparisons were conducted using the Ebayes method of the limma package. Nominal p-values are presented in volcano plot while corrected for multiple testing p-values with the Benjamini-Hochberg method are provided in Supplementary Data 2. c CTGF expression in the BVOs secretome, n = 5 BVOs per pooled sample, from 2 separate preparations. Statistical comparison was conducted using the Ebayes method of the limma package. Nominal p-value is displayed in beanplot while corrected for multiple testing p-value with the Benjamini-Hochberg method is provided in Supplementary Data 2. d QPCR quantification of CTGF expression in BVOs (n = 20 per group from three separate preparations) and in iPS-ECs (e) (n = 4 independent preparations). Data are shown as mean ± SD; p values were calculated using a two-tailed Student’s t-test (BVOs: **p = 0.0068, iPS-ECs: ****p = 0.000034). β-actin was used as a normalisation control. f Transcription factor enrichment analysis for differentially expressed proteins using the ChEA3 tool. Red circles indicate putative binding partners of YAP. g YAP reporter activity in HEK293T cells cultured as indicated for 24 h. rCTGF: recombinant CTGF. Data from three independent transfections in quadruplicates are shown as mean ± SD using two-way ANOVA followed by Tukey’s multiple comparisons tests. (CTR vs. PFK15 ****p = <0.000000000000001; CTR vs. PFK15 +rCTGF ****p = 0.000000002; CTR vs. rCTGF ****p = 0.000000013; PFK15 vs. PFK15+rCTGF **p = 0.0057; PFK15 vs. rCTGF **p = 0.0010). YAP subcellular localisation in iPS-EC after 3 h treatment with PFK15 as detected using (h) immunofluorescence confocal microscopy. N nucleus, C cytosol. n = 400 cells per group. Data are shown as mean ± SD using two-way ANOVA followed by Tukey’s multiple comparisons tests. (N > C: CTR vs. PFK15 ****p = 0.000000138; CTR vs. PFK15 +rCTGF ****p = 0.000064; CTR vs. rCTGF ****p = 0.000020; N = C: **p = 0.0030; ***p = 0.0001; ****p = 0.000000216) and (i) western blot analysis. Nucl nucleus, Cyto cytosol. Histone 3 expression (H-H3) was a normalisation control for the nuclear fraction. Loading control: Ponceau staining. Two independent experiments were performed. Source data are provided as a Source Data file.