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cAMP-dependent activation of protein kinase A attenuates respiratory syncytial virus-induced human airway epithelial barrier disruption

Fig 2

cAMP attenuates RSV-induced epithelial junctional disassembly via Epac-independent mechanisms.

(A,B) Confluent epithelial cell monolayers were infected with RSV at an MOI of 0.5 for 48 h in the presence or absence of a stable cAMP analog, 8-Bromo-cAMP (10–100 μM), or an Epac activator, 8CPT-2Me-cAMP (50–100 μM). TEER was measured at indicated time points. (C) Confluent airway epithelial cell monolayers were treated for 48 h with either vehicle, 8-Bromo-cAMP or 8CPT-2Me-cAMP. Cells were fixed and labeled for ZO-1 (green) and nuclei (blue). Scale bar, 40 μm. (D) Airway epithelial cell monolayers were infected with RSV (MOI, 0.5) for 48 h in the presence of the vehicle, forskolin (20 μM), 8-Bromo-cAMP (100 μM), or 8CPT-2Me-cAMP (100 μM). Cells were fixed and immunolabeled for different AJ (E-cadherin, β-catenin) and TJ (ZO-1, occludin) proteins. Note a marked TJ and AJ disassembly in RSV-infected, vehicle treated cells (short arrows) and preservation of normal AJC appearance in virus-infected cells treated with either forskolin or 8-Bromo-cAMP (long arrows). Scale bar, 40 μm. (E, F) Cells were infected with RSV in the presence of either vehicle, forskolin or cAMP analogs, followed by measuring TEER at the indicated times and transepithelial paracellular flux of FITC-dextran at 48 h of viral infection. (G) Immunofluorescence staining for claudin 1 and 4 in epithelial cells infected with RSV. There is marked TJ and AJ disassembly in RSV-infected cells and virus-infected cells treated with 8CPT-2Me-cAMP (short arrows). There is a preservation of normal AJC appearance in virus-infected cells treated with either forskolin or 8-Bromo-cAMP (long arrows). Scale bar, 40 μm. Each image and graph is representative of at least 3 independent experiments. Data is presented as mean ± SEM **, P< 0.01 and ***, P< 0.001 as compared to RSV-infected vehicle-treated cells.

Fig 2

doi: https://doi.org/10.1371/journal.pone.0181876.g002