Fig 1: Gating strategy and validation of immunophenotyping panel #2 (monocyte, dendritic cell (DC), natural killer (NK) cell panel). (A) The gating strategy was used to define selected cell populations. FSC versus SSC gates were initially used to separate monocytes and lymphocytes based upon the morphology. Cells were gated to obtain singlet cells. A viability dye was then used to exclude dead cells. The live cells were then gated with CD45. In the monocyte population, the CD45+ cells were then gated on CD3−, CD19− and CD14+ to obtain the CD14+ monocyte population. Activation status of these cells was determined by CD54/CD69 gates. In the lymphocyte population, the CD45+ cells were of each gated on CD3. CD3+ CD56+ cells indicated NK T cells. CD3− cell population was gated on CD19−, CD14−, CD20−. From this gate, the CD123+ gate was used to define pDC. The CD11c+ gate was used to define mDCs, and CD56+ was used to define NK cells. Activation status of each of these cell populations was defined with CD54/CD69. (B) Gating shown for unstimulated PBMC from one replicate of healthy donor I5F3. Red and blue overlays are the isotype controls while the black overlay is a replicate of the fully stained PBMC with labeling antibodies. Each gate is an inclusive gate (cells in gate are included in further gating) except the viability (live/dead) gate which is an exclusive gate (cells in gate are excluded from further gating). (C) PBMC from three healthy donors (N = 3, 2 technical replicates) were activated with positive controls (ODN2216/PHA-M) or left unstimulated/untreated for 24 h. The percentage of cells in each gate for each donor were determined following the same gating strategy as shown in A. The percentages indicate the percentage of selected cells in parent gate rather than in the total events. Each box plot represents data of 2 replicates from each of three donors (E3H8, O4Q7, I5F3). The middle bar is the median and the whiskers are the 10th and 90th percentiles of the data.
Fig 2: Coronary artery endothelial cell dysfunction markers are upregulated by IL-32 isoforms β and γ but not α. (A) Analysis of ICAM-I (CD54) and VCAM-I (CD106) RNA expression, normalized to the housekeeping gene β-glucuronidase, in primary CAEC showing their increased relative expression following 12 h stimulation with IL-32β and γ but not α (500 ng/mL, n = 6). (B) Analysis of ICAM-I and VCAM-I expression in CAEC supernatants following stimulation with IL-32α, β and γ for 72 h (n = 10). (C) Upper panels: Representative Flow cytometry data showing the gating strategy on CAEC live cells and overlapping histograms for ICAM-I and VCAM-I on the cell surface following stimulation with IL-32α, β and γ for 72 h (500 ng/mL). Lower panels: Analysis of ICAM-I and VCAM-I surface expression from n = 10 treatments (10 experimental replicates from CAEC). Data analyzed with the nonparametric test Kruskal-Wallis and Dunn’s subtest. NS: non-stimulated.
Fig 3: Pro-inflammatory stimuli promote the expression of adhesion molecules and mitochondrial ROS formation in male and female HUVECs. Male and female HUVECs were treated with TNF-α (10 ng/mL) or supernatant from pro-inflammatory THP-1 cells (1:1 dilution in cell culture medium) for 24 h. Real-time PCR analyses and flow cytometry (A,B) of VCAM-1 and (C,D) ICAM-1 and (E) mitochondrial ROS formation (MitoSOX assay). Data are presented as bar charts with individual data dots and means ± SEM (PCR: n = 8; FACS and MitoSOX n = 10). All data were normalized to the mean of the male basal control. Statistical analysis was performed using the paired Wilcoxon test. * p < 0.05, ** p < 0.01. basal: control; TNF-α: TNF-α treatment; THP-1 supernatant: THP-1 supernatant treatment.
Fig 4: Effects of resveratrol and metformin on adhesion molecule expression in TNF-α–activated male and female iPSC-EC. Flow cytometry analyses of (A) ICAM-1, (B) VCAM-1 and (C) E-selectin (CD62) mean fluorescence intensity (MFI) in male and female iPSC-EC under basal conditions and after TNF-α with or without resveratrol or metformin. Cells were stimulated with TNF-α (10 ng/ml, 24 h) and then treated with resveratrol (1 µg/ml) or metformin (1 µg/ml) for 24 h. Data are shown as bar charts with individual data dots and means ± SEM (n = 4–6 per group) and were normalized to the mean of the male basal control. Statistical comparisons were performed using non-parametric tests, and effects with p ≥ 0.05 are reported as non-significant trends.
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