FIGURE 7.

Increased NOS2 expression promotes E. coli proliferation. (A) RNA sequencing was used to determine the differentially expressed genes in mouse lungs infected for 48 h between individual infection and co-infection groups. All the genes whose expressions were upregulated and changed by at least 2-fold were displayed. The red arrow highlight the upregulated NOS2 genes. (B) The mRNA expression level of NOS2 in mouse lungs was examined by quantitative real-time PCR. (C) MLE12 cells were infected with or without H9N2 (MOI = 1) for 12 h, followed by E. coli (MOI = 10) infection with or without S-Methylisothiourea Sulfate (SMT) treatment. NO production in culture supernatants at the indicated time point was measured by NO assay kit. (D) MLE12 cells were infected with or without H9N2 as described in (C). OD₆₀₀ of E. coli in cell culture supernatants at the indicated time point was measured by spectrophotometer. (E) MLE12 cells were infected with or without H9N2 (MOI = 1) for 12 h, followed by E. coli (MOI = 10) infection with or without SMT treatment. After 6 h, the number of E. coli in cell culture supernatants was counted. (F) E. coli cultured in antibiotic-free DMEM was treated with SMT or vehicle control. OD₆₀₀ of E. coli in the supernatants at the indicated time point was measured by spectrophotometer. Data are presented as the mean ± SD of three independent experiments. **P < 0.01, ****P < 0.0001.