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. 2024 May 16;27(6):110011. doi: 10.1016/j.isci.2024.110011

Figure 1.

Figure 1

IFNγ/TNFα costimulation of HAECs induces nascent RNA production and synergistic expression of proinflammatory chemokine genes

(A) Schematic depicting treatment groups and experimental time points in HAECs.

(B) Schematic of experimental approach for MIMS experiments.

(C) MIMS images of HAEC stimulated with IFNγ/TNFα for 1 (top) or 4 h (bottom) versus unstimulated cells. 32S images reveal cellular and nuclear contours (left column). Hue saturation intensity images are used to visually represent isotope ratio measurements and in turn map 13C-thymidine labeling of DNA (13C/12C, middle) and 15N-uridine labeling of nascent RNA (15N/14N, right). The lower bound of the color scale (blue) is set to natural background (13C = 1.1%; 15N = 0.37%) and the upper bound of the scale is set to reveal differential labeling expressed as % above natural background. Scale bars = 5 μm. See also Figure S1B.

(D) Nuclear (top) and cytoplasmic (bottom) 15N-uridine labeling was quantified at the 1 and 4-h timepoints after stimulus and expressed as % above natural background. Each dot represents a different cell/nucleus with mean ± SD. One-way ANOVA, Dunnett’s test used for 4-h time point; Kruskal-Wallis/Dunn’s nonparametric test used for 1-h time point. ∗p < 0.05 ∗∗p < 0.01. Yellow dots correspond to the cells shown in the images in Figure 1C.

(E) Heatmap of averaged differential gene expression in unstimulated HAECs and HAECs treated with IFNγ (50 ng/mL), TNFα (25 ng/mL) or IFNγ/TNFα (n = 4 per condition).

(F) Scatterplot of difference in expression calculated by log2(FC) of IFNγ/TNFα (i.e., costimulated) minus the sum of the log2FC (IFNγ) plus log2FC (TNFα) (n = 4 per condition). Color indicates outlier status. All log2FC were calculated with cytokine vs. unstimulated cells.

(G) Scatterplot of ranked log2(FC) difference between IFNγ/TNFα minus the sum of the FC observed in IFNγ plus FC observed in TNFα. Green color indicates gene expression greater in IFNγ/TNFα, blue color indicates gene expression is less in IFNγ/TNFα than the sum of the FC observed in IFNγ plus FC in TNFα (n = 4 per condition).