Fig. 2

Females produce less amphiregulin (AREG) than males during influenza A virus infection. Using RNA isolated from lungs collected at either 7 dpi or 14 dpi, Areg mRNA expression was quantified and normalized to Gapdh using the ΔΔCt method (a n = 7–9/sex). Fixed lungs collected 14 dpi were used to assess AREG expression (in red) in pulmonary cells (DAPI+, in blue), and β-tubulin was used to identify ciliated epithelial cells (in green) using immunofluorescence (b). Representative images of bronchioles (× 10 magnification) and focused areas (× 40 magnification) are shown (b). The percentage of AREG+ cells in the lungs was analyzed and quantified using ImageJ (c n = 15 fields/sex). Mouse tracheal epithelial cell (mTEC) cultures were isolated and differentiated at an air liquid interface from male and female mice. Twenty-four hours after mock or 2009 H1N1 (MOI = 10) infection, Areg mRNA relative to Gapdh expression (ΔCt; d) and AREG (pg/ml) secretion into the basolateral media (e) were measured. Infectious virus titers in IAV-infected mTECs (f) were measured at the indicated time points (n = 8/sex/time point), with the dotted line indicated the TCID₅₀ limit of detection (LOD). Data represent means ± SEM from two independent experiments, with significant differences represented by asterisks (*)