Figure 4. NKX2–1^GFP;AGER^tdTomato reporter iPSC line enables tracking and purification of iAT1^YAP5SA cells.

(A) Gene editing strategy to generate BU3 NKX2–1^GFP;AGER^tdTomato (NGAT) dual reporter iPSC line for tracking lung epithelial lineages and AT1-like cells (BU3 NKX2–1^GFP reporter previously published by Hawkins et al.).⁵⁴

(B) AGER^tdTomato+ cells sorted and analyzed 14 days after transduction of iAT2s with lentiviral WT YAP or YAP5SA.

(C) Live cell fluorescence microscopy of YAP5SA-transduced cells growing next to an un-transduced epithelial sphere (GFP, tdTomato, and TagBFP fluorescence; scale bar, 200 μm).

(D) Flow cytometry of iAT2s (showing sorting gate for AGER^tdTomato + and − cells with AGER^tdTomato+ percentage quantified.

(E) Gene expression in sorted cells from (D). WT YAP, unsorted WT YAP transduced cells; YAP5SA-pre, unsorted YAP5SA transduced cells; TOM+,AGER^tdTomato+ sorted cells; TOM−, AGER^tdTomato−-sorted cells (N = 3 transductions, one-way ANOVA).

(F) Immunofluorescence staining of NKX2–1 protein and tdTomato (αRFP) (NKX2–1: green, F-actin: phalloidin white, tdTomato: red, nuclei: blue. Scale bars, 50 μm).

*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001, bars represent mean ± SD, BU3 NGAT iPSC line for all panels. See also Figure S3.