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. 2016 Jan 28;90(4):1788–1801. doi: 10.1128/JVI.02246-15

FIG 2.

FIG 2

Upregulation of GALNT3 mRNA by IAV replication. (A) Downregulation of miR-221 and miR-17-3p in A549 cells infected with PR8 virus. A549 cells were infected with PR8 virus at an MOI of 3.0, and the expression levels of miR-221 and miR-17-3p were monitored at the indicated time points by real-time RT-PCR. (B to E) IAV infection upregulates GALNT3 mRNA. (B) A549 and BEAS-2B cells were infected with H1N1 PR8 virus at an MOI of 3.0. A549 cells were infected with H1N1 Beijing (C), H3N2 Panama (D), and H3N2 Suita (E) viruses at an MOI of 3.0. The expression of GALNT3 was monitored by real-time PCR at the indicated times postinfection. Samples were normalized to GAPDH mRNA, and the relative expression levels of GALNT3 are shown (0 h = 1.0). (F) Upregulation of GALNT3 protein in bronchial and alveolar epithelial cells isolated from IAV-infected mice. After the intranasal infection of C57BL/6 mice with H1N1 PR8 virus at 103 PFU, the expression of the GALNT3 and IAV NP proteins in lung cells was detected by Western blotting. The line graphs indicate the relative band intensities of each protein compared to that of tubulin. The intensities of the bands were determined using a LAS-4000 imager (Fujifilm). (Left) GALNT3 protein; (right) IAV NP protein. (G) IAV infection does not change the mRNA expression levels of GALNT1, GALNT2, GALNT6, or GALNT7. A549 cells were infected with PR8 virus at an MOI of 3.0, and the mRNA expression levels of GALNT1, GALNT2, GALNT6, and GALNT7 were monitored at the indicated times by real-time RT-PCR. Samples were normalized to GAPDH mRNA, and the relative expression levels of GALNTs are shown (mock, 1.0). (H and I) Viral replication is necessary for the upregulation of GALNT3 mRNA. A549 cells were inoculated with UV-irradiated PR8 virus (UV-PR8) at an MOI of 3.0, and the replication of the inoculated virus was detected by immunofluorescence analysis using an anti-NP antibody at 12 h postinfection. Scale bar, 30 μm. The expression of GALNT3 mRNA was determined via real-time RT-PCR. All values are expressed as the means (n > 3) ± SEM. Statistical significance was analyzed by Student's t test. *, P < 0.05; **, P < 0.01.