Figure 4.

Lactate and RANTES enhance N protein-carried nucleic acid expression

(A) Lactate effect. 293T cells were pretreated with lactate at indicated concentrations. After treatment, N-GFP DNA mixtures were added to 293T cells. The GFP+ cells were counted after 24 h.

(B) Dose-dependent effect of RANTES. 293T cells were treated with an indicated dose of RANTES overnight. After treatment, Pre-mixed 10 μg mammalian cells-expressed WT N or Omicron N and 40 μg GFP-RNA (Left panel), GFP-DNA (Right panel) was added to cells overnight, and the number of GFP+ cells were counted under fluorescent microscopy.

(C) N-RNA and N-DNA complexes induced RANTES. HPAEpiC cells were stimulated by mammalian expressed N protein, GFP-RNA (or DNA), and N+GFP-RNA (or DNA) mixture for 24 h. After treatment, protein secretion of RANTES was detected by flow cytometry analysis.

(D) N protein was added to HPAEpic cells at indicated dosages for 30 min. After treatment, the cells were harvested and phosphorylation of p38 was detected by Western blotting. Quantification of band intensities was normalized by non-treated cells.

(E) 293T cells were pretreated by p38 inhibitor (SB203580) at indicated dosages overnight. After treatment, the N-GFP-DNA mixtures were added to 293T cells. GFP⁺ cells number were counted after 24 h.

(F) Mammalian cells-expressed N protein 4 μg pretreat with anti-N monoclonal antibody (NP-mAb-40 or 56) 10 μg/mL, and then mix with 12 μg GFP-DNA for 1 h, then the N+DNA/antibody complex are added to HPAEPic cells for 48 h. RANTES concentration in the supernatant is measured and detected by anti-RANTES beads by flow cytometry analysis. All data are shown as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; t test. See also Figures S6–S9.