Figure 2.

Validation of transcriptomic profiling of pulmonary endothelial cells (ECs). A, Experimental strategy for isolating endothelial-enriched transcripts in the lung. Cdh5-CreER^T2; RPL22^tm1.1Psam mice were maintained under normoxia as control group and Cdh5-CreER ^T2; Sox17^fl/fl RPL22^tm1.1Psam mice were exposed to hypoxia as the Sox17 ^i∆EC/hypoxic group following tamoxifen administration, which allowed the synthesis of hemagglutinin (HA)-tagged ribosomes specifically in ECs. Lungs were then harvested. B, Volcano plot of differentially expressed genes in lung ECs of control and Sox17^i∆EC/hypoxic mice. Sox17 highlighted in blue confirms its decreased level. C, Immunofluorescence image verifying EC-specific HA immunostaining in the lung. D, Fold changes in transcripts of marker genes for EC and other cell types induced by capturing HA-tagged ribosomes. Transcript levels in lung tissue samples for RNA sequencing are validated by quantitative PCR (n=3). Relative levels of transcripts are normalized to those of Gapdh. As controls, the averages of samples before the precipitation of HA-tagged ribosomes (left column of each pair) are arbitrarily set as 1. EC markers (Pecam1, Cdh5) are enriched, whereas markers of other cell types (Pdgfrb, pericytes; Acta2, VSMCs; Sftpc, pneumocytes) are excluded. Data are presented as mean±SD. E, Three-dimensional principal component (PC) analysis and (F) unsupervised clustering clearly separate RNA sequencing data from the control (n=4) and Sox17^i∆EC/hypoxic groups (n=4). Scale bar, 10 μm (C).