Fig 1.
SARS-CoV-2 spike protein inhibits ACE2 expression.
(A, B) Western blot analysis of SARS-CoV-2 spike protein expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with an empty vector or SARS-CoV-2 spike gene construct. (C, D) Western blot analysis of ACE2 and AT1 receptor expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. (E, F) Western blot analysis of ACE2 expression in Huh7.5 and A549 cell lysates prepared after 48 h of transfection of empty vector or SARS-CoV-2 spike S1 or S2 gene construct. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.
Fig 2.
SARS-CoV-2 spike protein activates the transcription factors for IL-6 synthesis.
(A, B) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in Huh7.5 and A649 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. (C, D) Western blot analysis of phospho-NF-κB (Ser276), IκBα and c-Fos expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.
Fig 3.
Candesartan cilexetil as an AT1 receptor antagonist prevent MAPK activation.
(A) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in Huh7.5 cell lysates prepared after 48 h of transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. Expression level of actin in each lane is shown as a total protein loading control for comparison. (B) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in A549 cell lysates prepared after 48 h of transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.
Fig 4.
SARS-CoV-2 spike protein stimulates IL-6 and soluble IL-6R production.
(A) The extra-cellular level of IL-6 was measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. (B) The extra-cellular level of soluble IL-6R was similarly measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment as shown in panel A. The results are presented as means ± standard deviations. ‘*’ and ‘**’ represent statistical significance p<0.05 and p<0.005, respectively. (C) The level of IL-6 was measured by ELISA from the serum samples of SARS-CoV-2 infected patients (n = 20) and uninfected healthy volunteers (n = 8). (D) The level of soluble IL-6R was similarly measured by ELISA from the serum samples of SARS-CoV-2 infected patients (n = 20) and uninfected healthy volunteers (n = 8) as shown in panel C. The results are presented as means ± standard deviations. ‘*’ and ‘**’ represent statistical significance p<0.05 and p<0.005, respectively. (E, F) Western blot analysis of ADAM-17 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or infected with SARS-CoV-2 virus, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. Expression level of actin in each lane is shown as a total protein loading control for comparison.
Fig 5.
SARS-CoV-2 spike protein regulates ADAM-17 activity.
(A) ADAM-17 enzymatic activity was measured from crude extracts of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene constructs. (B) ADAM-17 enzymatic activity was measured from crude extracts of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike S1 or S2 gene construct or empty vector. The results are presented as mean ± standard deviation. ‘*’ represent statistical significance (p<0.05). (C, D) Western blot analysis of ACE2 expression in Huh7.5 and A549 cell lysates prepared after 48 h of transfection of empty vector or SARS-CoV-2 spike S1 gene construct in the presence or absence of ADAM-17 inhibitor. Expression level of actin in each lane from same gel is shown as a total protein loading control for comparison.
Fig 6.
SARS-CoV-2 spike protein causes inhibition of tyrosine phosphorylation of STAT3.
(A, B) Western blot analysis of phospho-STAT3 (Tyr705) and total STAT3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transient transfection of SARS-CoV-2 spike gene constructs or infection with SARS-CoV-2 virus. (C, D) Western blot analysis of phospho-STAT3 (Ser727) and total STAT3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transiently transfection of SARS-CoV-2 spike gene construct or infection with SARS-CoV-2 virus. (E, F) Western blot analysis of SOCS3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transiently transfected with SARS-CoV-2 spike constructs or infected with SARS-CoV-2 virus. Expression level of actin in each lane from the same gel is shown as a total protein loading control for comparison.
Fig 7.
IL-6 trans-signaling induces MCP-1 expression.
(A, B) Western blot analysis of MCP-1 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or infected with SARS-CoV-2 virus. (C) Western blot analysis for IL-6Rα expression in Huh7.5, A549, or TMNK-1 cell lysates. (D) Western blot analysis of phospho-STAT3 (Tyr705) and MCP-1 expression in TMNK-1 liver endothelial cell lysates prepared after treated with or without culture supernatant from SARS-CoV-2 spike gene expressed A549 cells in presence or absence of Candesartan cilexetil and LPS. (E) Western blot analysis for phospho-STAT3 (Tyr705) and MCP-1 expression status in TMNK-1 liver endothelial cell lysates prepared after treatment with LPS and culture supernatant from SARS-CoV-2 spike gene expressed A549 cells in the presence or absence of Tocilizumab. Expression level of actin in each lane from the same gel is shown as a total protein load for comparison. (F) The extra-cellular level of MCP-1 was measured by ELISA in culture supernatant of TMNK-1 cells after treatment with LPS alone or together with culture supernatant from SARS-CoV-2 spike gene expressing A549 cells in the presence or absence of Tocilizumab. The MCP-1 expression level in the culture supernatant from SARS-CoV-2 spike gene expressing A549 cells was also detected. (G) Comparative analysis of cellular migration of human monocytes (THP-1) in the presence of culture supernatant from TMNK-1 cells treated with LPS, and from culture supernatant of SARS-CoV-2 spike gene expressed A549 cells in the presence or absence of Tocilizumab. The results are presented as mean ± standard deviation. ‘*’ (p<0.05) and ‘**’ (p<0.005) represent statistical significance.
Fig 8.
SARS-CoV-2 spike protein induces alarmin secretion from epithelial cells.
(A, B) The extra-cellular level of IL-1α and HMGB1 were measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct or empty vector as a control for comparison. The results are presented as mean ± standard deviation. ‘*’ (p<0.05) and ‘**’ (p<0.005) represent statistical significance.
Fig 9.
(A, B) Overview of IL-6 mediated classical and trans-signaling mechanisms. (C) Schematic presentation of molecular changes occurring upon SARS-CoV-2 infection or spike protein expression in epithelial cells.