Fig. 5.

Responses to SARS-CoV-2 components and treatment with CO₂ in primary ECs. ERK1/2 activity was determined by immunoblotting with an anti-phospho-ERK1/2 antibody, and protein loading was assessed by Ponceau S staining and immunoblotting with anti-GAPDH and anti-ERK1/2 antibodies. Changes in the expression of specific proteins were detected with anti-HIF-1α and anti-ICAM-1 antibodies. IL-6 level was measured by ELISA. The means ± SDs of three independent experiments are presented. a The scheme of the course of experiments. In b–g, ECs were incubated with mock or the indicated SARS-CoV-2 derivative for 22 h. Then, 8.7% CO₂ was applied for 12 min. Cells were frozen 24 h after the start of the experiment. In h–l, ECs were incubated with mock or the indicated SARS-CoV-2 derivative for 20 h. Then, 7.5 ng/ml IFNγ was added. After 2 h, treatment with 8.7% CO₂ for 12 min was conducted. Cells were collected 24 h after the beginning of the experiment. b 50 ng/ml spike, HUVECs. c Heat-inactivated SARS-CoV-2, USA-WA1/2020 isolate, HUVECs. A number of administered viral particles corresponded to the multiplicity of infection (MOI) of 7.5. d Spike-pseudotyped lentivirus, HUVECs. e 50 ng/ml spike, HPMECs. f Heat-inactivated SARS-CoV-2, USA/CA_CDC_5574/2020 isolate, MOI 3, HPMECs. g Spike^D614G-pseudotyped lentivirus, HPMECs. h 50 ng/ml spike with 7.5 ng/ml IFNγ, HPMECs. i Heat-inactivated SARS-CoV-2, USA/CA_CDC_5574/2020 isolate with 7.5 ng/ml IFNγ, HPMECs. j Spike^D614G-pseudotyped lentivirus with 7.5 ng/ml IFNγ in HPMECs. k 50 ng/ml spike with 7.5 ng/ml IFNγ, HUVECs. l HUVECs were incubated with mock or the indicated SARS-CoV-2 derivative for 18 h 45 min. Then, 7.5 ng/ml IFNγ and 10 ng/ml TNFα were added. After 15 min and subsequently after 3 h 15 min, the cells were treated with 8.7% CO₂ for 12 min. Cells were collected 24 h after the beginning of the experiment. pERK1/2—phosphorylated ERK1/2