RNA-seq of cultured lung epithelial cells from P6 wild-type mice with and without Piericidin A

In order to evaluate the cell autonomy of the phenotypes observed in NDUFS2 cKO mice, we performed in vitro studies with culture media supplemented with aspartate and asparagine. Postnatal transitional cells normally start to appear from P7. Therefore, to evaluate early postnatal transcriptomic signatures, we performed bulk RNA-sequencing of lung epithelial cells isolated from NDUFS2 cKO and control mice at P6. The expression of ISR genes was slightly higher in NDUFS2 cKO lungs at P6 compared to NDUFS2 control lungs, but not as high as P35 NDUFS2 cKO lungs. Moreover, transcriptomic signatures of control and cKO lung epithelial cells at P6 were not clearly separated in principal component analysis (PCA), suggesting the critical pathways are disrupted after P6. We then isolated lung epithelial cells from P6 wild-type mice, before transitional cells appear, and cultured them on 2-D plastic culture plates, a system in which AT2 cells have long been recognized to spontaneously differentiate into cells resembling AT1 cells. Using RNA-sequencing, we confirmed that AT2 cells lose AT2 cell markers and express AT1 cell markers in this system 72 hours after isolation. However, adding mitochondrial complex I inhibitor Piericidin A to the culture media prevented AT2 cells from expressing AT1 cell markers likely through the high ISR activation