Fig 2: Lung ischemia-reperfusion experimental procedures. (A) Animals were randomized into five groups: the SHAM group, SHAM + DIZE group, IR group, IR + ACE2-inhibited group, and IR + DIZE group. For the IR + DIZE and SHAM + DIZE groups, 0.1% DIZE 15 mg/kg/day was injected intreperitoneally. For the IR + ACE2-inhibited group, a potent ACE2 inhibitor, MLN-4760 was injected via the caudal vein before surgery. For the IR group, IR + ACE2-inhibited group and IR + DIZE group, a lung IR injury model were established. For the SHAM and SHAM + DIZE groups, only tracheotomy, mechanical ventilation and open chest operation were performed. (B) The key procedure during the surgery was to find the bronchus accompanied by the pulmonary artery. (C) The lung deflated when the hilum was clamped.

Fig 3: Overexpressed WT ACE2 subcellular localization and stability. A Immunofluorescence confocal imaging of permeabilized HeLa cells transfected with Flag-tagged ACE2 (red), GFP-tagged HRas (plasma membrane marker in green) and Calnexin (ER marker in blue). Images were acquired using 100X magnification and manipulated by ImageJ. Scale bar = 50 µm. B HEK293 cells transiently transfected with GFP and Flag-tagged WT ACE2 plasmids for 48 Hrs. Lysates were then digested in presence or absence of Endo H and PNGase F enzymes for 3 Hrs and 1 Hrs, respectively. Anti-Flag primary antibody was used to stain WT ACE2 and anti-GFP antibody was used to satin GFP that was used as a transfection control. C Flag-tagged WT ACE2 transfected cells were treated with DMSO and 100 µg/ml cycloheximide for a period of 18 Hrs. Cells were lysed at the indicated time points (0, 4, 8, 12, and 18 Hrs) and lysates were analyzed by western blotting. Anti-Flag antibody was used to stain WT ACE2. Mock sample represent un-transfected cells. In all the experiments actin was used as a loading control. D Graph representing the relative expression of WT ACE2 compared to DMSO treated cells at the indicated time points. Error bars represent ± SEM of three independent experiments. Band quantification was performed using ImageJ

Fig 4: Infection of brain organoids with pseudotyped SARS-CoV-2. (A) Schematic illustration of pseudotyped SARS-CoV-2 infection of brain organoids and cortical neurons. (B) Immunofluorescence of mock-treated or SARS-CoV-2 pseudovirus-treated dorsal forebrain organoids (6 months) for detecting pseudotyped SARS-CoV-2 spike (mCherry) and TUJ1. (C) Immunofluorescence of mock-treated or SARS-CoV-2 pseudovirus-treated dorsal forebrain organoids (6 months) for detecting pseudotyped SARS-CoV-2 spike (mCherry) and ACE2. (D) Quantification of immunofluorescence indicates the percentage of mCherry-positive cells among the total DAPI-positive cells. In D, data are presented as mean ± SD (n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar, 100 µm.

Fig 5: Correlation between protein levels of ACE2/ACE and apoptotic factors during an APE. ACE, angiotensin-converting enzyme; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein.