Fig. 6. SARS-CoV-2 N globally alters cytokine secretion and triggers endothelial barrier breakdown in a variant-specific manner.

(A) Heatmap showing TLR7/8-induced cytokine responses across THP-1 macrophage-like cell lines expressing none, Wuhan-Hu-1, Delta, or Omicron BA.1 N proteins. Values represent mean log₂ fold change relative to untreated controls. Cytokines shown are the top 25 most modulated relative to parental cells (Wilcoxon test; asterisks indicate significant differences compared to parental cells: *P < 0.05; nonsignificant differences are not shown). (B and C) Global cytokine responses to TLR7 stimulation. Cytokines significantly induced (B) or suppressed (C) by TLR7/8 stimulation in parental THP-1 macrophage-like cells were used as fixed reference sets to assess how N variants modulate cytokine programs. Values represent log₂ fold change relative to untreated controls. Each point represents one cytokine. Box plots show the distribution of cytokine responses across parental and variant N-expressing cells (paired Wilcoxon test; asterisks indicate significant differences compared to parental cells: *P < 0.05; **P < 0.01; nonsignificant differences are not shown). (D) Schematic of THP-1 macrophage-like cell-conditioned medium experiment. This figure was created using BioRender.com with the following credit: Created in BioRender. Li, M. (2026) https://BioRender.com/0u1valk. (E and F) Fold change in TEER measurements in HCAECs (E) and BMECs (F) treated with conditioned medium from the indicated cell lines. Data shown are fold changes in TEER observed 24 hours post-treatment, relative to 0 hours post-treatment. TNF-α was included as a positive control for barrier disruption. All graphs show mean ± SD. Data shown are combined from two independent experiments (asterisks indicate statistically significant differences; two-way ANOVA and Šidák’s multiple comparisons: *P < 0.05; **P < 0.001; ****P < 0.0001; nonsignificant differences are not shown).