Fig. 5

Functional assays reveal that FOXI1 KO leads to decreased numbers of ciliated cells in hiPSC-AECs. A: Airway surface liquid (ASL) pH of mature FOXI1 WT and KO hiPSC-AECs. Filled circles represent individual values and bars are means ± SD (n = 6 consists of 3 independent experiments, 2 biological replicates per experiment); Mann-Whitney test. B: Transepithelial resistance (R_t) of mature FOXI1 WT and KO hiPSC-AEC ALI cultures. Filled circles represent the average of 3 technical replicates (measurements) and bars are means ± SD (n = 6 ALIs from 3 independent experiments, 2 biological replicates per experiment). * P < 0.05; Mann-Whitney test. C: Ciliary beat frequency (CBF) of FOXI1 WT and KO hiPSC-AEC ALI cultures. Filled circles represent the average of values obtained from 5–20 FOVs with > 5% of coverage from one sample and bars are means ± SD (n = 3 independent experiments); Student’s t-test. D: Area covered with motile cilia in FOXI1 WT and KO hiPSC-AEC ALI cultures. Filled circles represent the average of up to 20 FOVs from one sample and bars are means ± SD (n = 3 independent experiments); Student’s t-test. E: Flow cytometry analysis of the amount of FOXJ1 + ciliated cells in FOXI1 WT and KO hiPSC-AEC ALI cultures. Gating was performed compared to stained hiPSC controls. Filled circles represent individual values and bars are means ± SD (n = 4 independent experiments); *P < 0.05; Mann-Whitney test. F: Cropped representative Western blot images show the expression of the ciliated cell marker DNAI1 in mature FOXI1 WT and KO hiPSC-AECs. Primary basal cells and HBEC ALI cultures served as negative and positive controls, respectively. Vinculin served as a loading control. G: Representative immunofluorescence staining of FOXJ1, acetylated tubulin (AcTub) and DAPI in FOXI1 WT and KO hiPSC-AECs. The scale bar is 100 μm