Figure 2.

BLM induces morphological change in epithelial cells in FD-AOs

(A) Live-cell imaging of epithelial cells in BLM-treated FD-AOs. Scale bars, 100 μm.

(B and C) Quantification of diameter and thickness of alveolar spheroids. Data are presented as mean ± SEM (n = 140 spheroids from 7 independent experiments). Unpaired two-tailed Student’s t test: ^∗∗∗p < 0.001.

(D) Expression levels of AT2 (SFTPC, ABCA3) and AT1 (AQP5, AGER) markers in FD-AOs evaluated by qRT-PCR. Gene expression in the normalizer (adult lung RNA control) was set at 1. Data are presented as mean ± SEM (n = 4). Unpaired two-tailed Student’s t test: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; ns, not significant.

(E) IFA of FD-AOs. Green, SPC-GFP; red, AGER; blue, nuclei (Hoechst). Scale bars, 20 μm.

(F) Total number of EpCAM⁺ or EpCAM⁻ cells in a well. All dissociated cells were counted and multiplied by the ratio of EpCAM⁺ to EpCAM⁻ cells as quantified by FCM. Data are mean ± SEM (n = 6 independent experiments). Two-way ANOVA with Sidak’s multiple comparisons test: ^∗∗∗p < 0.001; ns, not significant.

(G) FCM of DMSO- and BLM-treated FD-AOs.

(H) Analyses of cell component ratio of EpCAM⁺ cells in FD-AOs using FCM. Data are presented as mean ± SEM (n = 5 independent experiments). Unpaired two-tailed Student’s t test: ns, not significant.

(I) Transmission electron microscopy images of BLM-treated FD-AOs. Scale bars, 2 μm.

(J) Mean fluorescence intensity of LysoTracker in FCM. Data are presented as mean ± SEM (n = 5 independent experiments). Two-way ANOVA with Sidak’s multiple comparisons test: ^∗∗p < 0.01; ns, not significant.