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. 2021 Nov 19;24(12):103478. doi: 10.1016/j.isci.2021.103478

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© 2021 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Panx-1 channel opening is essential for SARS-CoV-2 replication

Immunofluorescence staining was performed on re-sectioned human lung tissue biopsied from patients with COVID-19 or tumors biopsied from patients with lung cancer as a control. Confocal microscopy was conducted to determine the expression and distribution of Panx-1 and other cellular markers. Tissues were stained for DAPI (a nuclear marker, blue), Panx-1 (green), EpCam (an epithelial cell marker, red) or Iba-1 (a macrophage marker, not shown), and SARSsense (a probe for SARS-CoV-2 mRNA using the RNAscope V-nCoV2019-sense probe).

(A) Staining of uninfected tumor lung tissue for DAPI, Panx-1, EpCam, and SARSsense at 10× magnification and (B) 40× magnification (n = 3). No significant levels of SARSsense staining for viral mRNA were detected. Note that EpCam and Panx-1 are expressed at the borders of an intact alveolar wall.

(C) Staining of lung tissue from patients infected with SARS-CoV-2 for DAPI, Panx-1, EpCam, and SARSsense at 10× magnification and (D) 40× magnification (n = 6). Low but significant levels of SARSsense staining for viral mRNA were detected. Significant levels of EpCam and Panx-1 expression were detected, with expressions elevated near the alveolar wall. Note that for this sample, the morphological integrity of the alveolar wall is retained.

(E) Staining of lung tissue from patients infected with SARS-CoV-2 for DAPI, Panx-1, EpCam, and SARSsense at 10× magnification and (F) 40× magnification (n = 5). Significant levels of SARSsense staining for viral mRNA were detected. Decreased but significant levels of EpCam and Panx-1 were detected. Note that for this sample, the morphological integrity of the alveolar wall has been compromised (represented by the dashed circle).

(G) Quantification of the total pixels per area for the control (C) and COVID-19 (CV)-infected conditions was low but significant for EpCam (CV, p = 0.00025, n = 9 cases with 6 sections per individual). In contrast, Panx-1 expression was high and significant (CV, p = 0.00153, n = 9 cases with 6 sections per individual). SARSsense (Sense) staining for mRNA was low but significant.

(H) Quantification of the total pixels per area for the control (C) and COVID-19 (CV)-infected conditions for Iba-1 was high and significant (p = 0.0031, n = 9, not shown). Similarly, Panx-1 expression was high and significant (p = 0.00102, n = 9). SARSsense (Sense) staining for mRNA was again low but significant.

(I) Quantification of the Pearson's colocalization index between EpCam, Iba-1, and RBC with Panx-1 in the control (C) and COVID-19 (CV)-infected conditions. EpCam with Panx-1 staining in COVID-19-infected patients was decreased relative to the control and indicated progressive deterioration of the alveolar wall (p = 0.001, n = 9). In contrast, Iba-1 with Panx-1 staining in COVID-19-infected patients was ∼2-fold higher relative to the control and indicated an increase in expression of Panx-1 in macrophages (p = 0.001, n = 9). Similarly, red blood cell (RBC) hemoglobin autofluorescence to Panx-1 staining indicates that the expression of Panx-1 increased in red blood cells of COVID-19-infected patients (p = 0.0025, n = 9).