Fig 2: IL‐1β&OxPAPC and siRNA standardisation by immunofluorescence A–DRepresentative fluorescent micrographs (Scale bar = 100 μm) and quantification of (A) ICAM1 intensity, (B) CM‐H2DCFDA (ROS indicator) intensity, (C) VE‐cadherin intensity and (D) nuclei (DAPI) number in HAEC transfected with Scrambled siRNA or siRNA to ICAM1, CDH5 and PLK1, and treated or not with IL‐1β and OxPAPC. The data are represented as a Z‐score (Materials and Methods). The experiment was repeated 5 times independently (biological replicates), with 4–5 technical replicates from each condition. The representative images of DAPI staining of Ctrl Scrambled in (C) are the same as those used in (A). Source data are available online for this figure.

Fig 3: siRNA library high content screen data AHeatmap summarising the outcome of the immunofluorescence assays for ICAM1 expression (antibody anti‐ICAM1), ROS intensity (probe CM‐H2DCFDA), VE‐cadherin expression (antibody anti‐VE‐cadherin) and nuclei counts (DAPI). Colour reflects the median Z‐score from all replicates.B–EQuantification of (B) nuclei (DAPI) counts, (C) ICAM1 intensity, (D) ROS (probe CM‐H2DCFDA) intensity and (E) VE‐cadherin intensity in HAEC transfected with Scrambled siRNA or siRNA to 81 target genes, and treated with IL‐1β and OxPAPC. The data are represented as a Z‐score (Materials and Methods). All experiments were repeated 5 times independently (biological replicates), with 8 technical replicates for all controls in the plate each time. P‐values were computed by multiple pairwise comparisons with the Wilcoxon test followed by BH correction. The comparisons were made between HAECs transfected with each of the 81 siRNA versus HAEC transfected with Scrambled siRNA and treated with IL‐1β and OxPAPC. Bootstrapping analysis was used to mitigate the unbalanced Scrambled_IL1B&OxPAPC control versus the 81 siRNA‐targeted genes (Materials and Methods). The boxplot depicts the median within the 25th and 75th percentiles, which the whisker extends no further than 1.5 × IQR (Interquartile Range).FAggregated rank analysis for genes exacerbating (red) and ameliorating (blue) endothelial dysfunction (ED) phenotypes upon knockdown. The first ranking, from left to right, rates genes by Z‐scores, with increased ICAM1 and ROS and decreased DAPI and VE‐cadherin. These are pro‐ED genes. The second ranking is for anti‐ED genes, with opposite criteria (Materials and Methods). Both are displayed on the same plot, represented by the 2 dots per gene. Source data are available online for this figure.

Fig 4: Drug treatment high content screen data A, B(A) Representative fluorescent micrographs (Scale bar = 50 μm) and (B) quantification of percentage of cell death calculated from a cell viability assay (calcein and ethidium staining) in HAEC treated with the 17 drugs at 10−8 and 10−6 M, without IL‐1β and OxPAPC. Starving treatment for 24 h was used as a cell death positive control. Data are presented as mean ± standard deviation. The experiment was repeated 3 times independently (biological replicates), with 1 technical replicate from each condition. P‐values were computed by multiple pairwise comparisons with the Wilcoxon test followed by BH correction. The conditions were compared to starving.C–FQuantification of (C) ICAM1 intensity, (D) CM‐H2DCFDA (ROS indicator) intensity, (E) VE‐cadherin intensity and, (F) nuclei (DAPI) number in HAEC treated with IL‐1β and OxPAPC alone (DMSO solvent), or in combination with inhibitor drugs at 10−6 M or 10−8 M. The data are represented as a Z‐score (Materials and Methods). All experiments were repeated 2 times independently (biological replicates), with 4–5 technical replicates from each condition. Only significant results (P‐value < 0.05) are shown. The boxplot depicts the median within the 25th and 75th percentiles, which the whisker extends no further than 1.5 × IQR (Interquartile Range).GAggregated rank analysis combining the siRNA and drugs screening exacerbating (red) and reducing (blue) ED phenotypes upon treatment. The first ranking, from left to right, rates genes by pro‐ED ranking scores (Figs 2F and 4B). These are pro‐ED genes. The second ranking is for anti‐ED genes, with anti‐ED ranking score (Materials and Methods). Both are displayed on the same plot, represented by the 2 dots per target. Source data are available online for this figure.

Fig 5: siRNA transfection and immunofluorescence APercentage of cell death calculated from a cell viability assay (calcein and ethidium staining) in HAEC transfected with Scrambled siRNA or siRNA to 81 genes, without treatment with IL‐1β and OxPAPC. P‐values were computed by multiple pairwise comparisons with the Wilcoxon test followed by BH correction. Data are presented as mean ± standard deviation. The boxplot depicts the median within the 25th and 75th percentiles, which the whisker extends no further than 1.5 × IQR (Interquartile Range). The experiment was repeated 4 times independently (biological replicates), with 8 technical replicates for all controls in the plate each time. All the comparisons were made between HAECs transfected with each of the 81 siRNA versus HAEC transfected with Scrambled siRNA.B–DRepresentative fluorescent micrographs (Scale bar = 100 μm) of (A) ICAM1, (B) CM‐H2DCFDA (ROS indicator) and (C) VE‐cadherin in HAEC transfected with Scrambled siRNA or siRNA to JUNB and TRAF1, and treated or not with IL‐1β and OxPAPC. The representative images of DAPI and CM‐H2DCFDA staining of Scrambled IL1B&OxPAPC in (C) are the same as those used in Fig EV2B. Source data are available online for this figure.