Extended Data Fig. 4: Human airway model exposes distinct steps of P. aeruginosa tissue colonization.

a. Images of the lung tissue surface before and after washing inserts with HBSS shows efficient mucus removal. Staining: mucus: Jacalin-fluorescein (green), membrane: CellMask DeepRed (purple). b. P. aeruginosa growth on the apical side of the lung tissues (continuous line) and spread to the basal compartment after tissue breaching (dotted line) with (black) and without (blue) apical mucus upon infection. c. Epithelial barrier integrity (TEER, plain lines) and tissue viability (LDH release, dashed lines) during infection in absence (blue) or presence (black) of mucus. d. Stills of live imaging (25×) of apoptotic events upon infection with P. aeruginosa. Staining: apoptotic cells: NucView (caspase-3/7 activity), membrane: CellMask DeepRed. e. Quantification of dead epithelial cells by FACS, using a viability marker (Zombie NIR), upon infection with P. aeruginosa. Unpaired t test (two-tailed) compared to uninfected; ** P = 0.0011. f. P. aeruginosa population on the airway epithelium (continuous line) and in the basal compartment (dashed line) with PneumaCult-ALI culture medium (black) or HBSS buffer (green) in the basolateral compartment. g, h, i. P. aeruginosa population on the apical (g) and basal side (h) of the lung tissues as well as tissue viability (LDH release) (i) upon infection with different inoculum sizes as indicated. For panels a-i: same donor. j, k. Comparison for 3 different donors of P. aeruginosa growth on the apical side of the lung tissues (continuous line) and spread to the basal compartment after tissue breaching (dotted line) with (black) (j) as well as tissue viability (LDH release) (k). For panels a-f, j, k: inoculum = 10³. For all panels n = 3 independent experiments. For j and k: data are presented as mean values +/− SD.