Fig 1.
Schematic diagram of primary human bronchial epithelial cell isolation and cultivation.
(A) Isolation procedure for primary human bronchial epithelial cells (B) Primary human bronchial epithelial cells cultured under submerged (left) and air-liquid interface conditions (right).
Fig 2.
Schematic diagram of the infection and experimental procedure of differentiated human bronchial epithelial cells.
Fig 3.
Efficient apical infection of differentiated human bronchial epithelial cells with HAdV-B14p1.
(A) Intracellular HAdV genomes were quantified by qPCR at 1 h and 48 h p.i. (** p< 0.01; *** p< 0.001, unpaired t-test) (B) Release of infectious virus progeny at the apical side of the differentiated bronchial epithelial cell layer as determined by the TCID50 method on day 1, 4, and 8 p.i. (** p< 0.01; *** p< 0.001, unpaired t-test). (C) Release of infectious virus progeny at the basal side of the differentiated bronchial epithelial cell layer as determined by the TCID50 method on day 1, 4, and 8 p.i. (* p< 0.05; *** p< 0.001, unpaired t-test). The TCID50 values in B and C are normalized against the input virus titers measured on day 1 p.i. and set to 1 x 100 because the TCID50 values measured on day 1 are probably remaining viral particles originating from the virus inoculum.
Fig 4.
Immunofluorescence staining of differentiated human bronchial epithelial cells.
Confocal microscopy immunofluorescence analysis of differentiated human bronchial epithelial cells. Figs A, B, C show XY planes, Figs D, E, F show XZ planes. (A, D) Cells were infected from the apical side with HAdV-B14p1 at a moi of 10 (TCID50/cell), fixated 4 days p.i. and stained for HAdV in green (FITC conjugated antibody) and the desmoglein 2 (DSG2 receptor) in red (dsRed antibody), the nucleus was counterstained in blue (DAPI). (B, E) Differentiated human bronchial epithelial cells stained for DSG2 receptor in red (dsRed) and occludin (OCLN) a tight junction marker in green (FITC), nucleus was counterstained in blue (DAPI). (C, F) Differentiated human bronchial epithelial cells were stained for CAR receptor in red (dsRed) and the tight junction marker OCLN in green (FITC) and the nucleus in blue (DAPI).
Fig 5.
Induction of chemokines after apical HAdV infection of differentiated human bronchial epithelial cells.
(A) IP-10 concentration in cell culture medium on day 4 and 8 p.i. as determined by ELISA (B) I-Tac concentration in cell culture medium on day 4 and 8 p.i. as determined by ELISA (n.s.: not significant, * p< 0.05; *** p< 0.001, two way ANOVA). Values shown are SEM values of quadruplicate infections.