Immunofluorescent staining of lung tissue from tamoxifen-treated SS chimeras shows knockdown of SMC CYB5R3 in pulmonary arteries of SS/R3^KDanimals. Representative immunofluorescent images of postmortem lung tissue from SS/R3^WT and SS/R3^KD chimeras showing WT (left) and knockdown (right) levels of CYB5R3 at 3 weeks (A) and 12 weeks (D) following completion of tamoxifen-induced Cyb5r3 KO. A Nikon A1 confocal laser microscope was used to image the pulmonary arteries at ×40 magnification with 1096 × 1096 resolution. Z-stack imaging (1-µm increments) of stained and immunoglobulin G control sections were used for the maximum intensity projection representative images. Smooth muscle α actin (green), CYB5R3 (red), PECAM (gray), and DAPI-stained nuclei (blue). Quantification of Cyb5R3⁺ per ACTA2⁺ (smooth muscle) area in SS/R3^WT (red bar) and SS/R3^KD (blue bar) chimeras completing 3- (B) and 12-week (E) studies. Quantification of Cyb5R3⁺ per PECAM⁺ (endothelial) area in SS/R3^WT and SS/R3^KD chimeras completing 3- (n = 9-11) (C) and 12-week studies (n = 5 each) (F). Regions of interest were drawn onto the maximum intensity projection for ACTA2 (SMC), which was then superimposed on the maximum intensity projection for CYB5R3 using ImageJ software. The quantity of CYB5R3 staining in medial smooth muscle and endothelium was determined via ImageJ using raw integrated intensity per area. The mean ± SEM is represented. The Student unpaired t test was used to determine statistical significance.