Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hypercapnia Suppresses Macrophage Antiviral Activity and Increases Mortality of Influenza A Infection via Akt1

doi: 10.4049/jimmunol.2000085

Figure Lengend Snippet: AMØs obtained by BAL from untreated mice were immunostained with isoform-specific antibodies for Akt1 (red), Akt2 (white) and Akt3 (green); nuclei were stained with DAPI (blue), n=5, results representative of at least 3 independent experiments (A). Differentiated THP-1 MØ were exposed to 5% CO2 (NC) or 15% CO2 (HC) in the absence or presence of specific inhibitors of Akt1 or Akt2, A674563 (50 nM) and CCT128930 (1 μM), respectively, and infected with IAV (MOI 2). Expression of RIG-I (B) and viperin (C) protein was assessed by immunoblot, and viral titers in culture supernatants were assessed by plaque assay (D); mean ± SEM, n = 4 from 4 independent experiments, *p<0.05 vs NC NI; **p<0.05 vs NC + IAV; ***p<0.05 vs HC + IAV. In additional experiments, mice were infected intranasally with lentivirus containing non-silencing (NS) shRNA or Akt1-targeted shRNA, and 21 days later AMØs were collected by BAL and immunostained for Akt1 (red) and F4/80 (green, MØ marker) (E) or lysed to measure Akt1 expression by immunoblot (F), n=4, results representative of at least 2 independent experiments. Alternatively, AMØs from the shRNA-treated animals were cultured in NC or HC, infected with IAV (MOI 2), and 18 h later processed for quantitation of viral NS1 protein by immunoblot (G); n=4, results representative of at least 2 independent experiments, *p<0.05 vs NS shRNA, ##p<0.05 vs NS shRNA + IAV in NC, ### p<0.05 vs NS shRNA + IAV in HC.

Article Snippet: The pLKO vectors either encoded two specific shRNAs against mouse Akt1 (Cat# TRCN0000304683) or a non-silencing control shRNA sequence (Cat# SHC002) (all from Sigma).

Techniques: Staining, Infection, Expressing, Western Blot, Plaque Assay, shRNA, Marker, Cell Culture, Quantitation Assay