Fig. 2

The activation of Cx43 hemichannels induced by spike S1 is potentiated by ACE2. (A) Averaged DAPI uptake rate by HeLa-parental, HeLa-Cx43^GFP, HeLa-Cx43^GFP/ACE2^mCherry or HeLa-ACE2^mCherry cells bathed with normal saline (control, white bars) or a divalent cation-free solution (DCFS, yellow bars). ***p < 0.001; effect of DCFS compared to control (two-way ANOVA followed by Tukey’s post-hoc test). (B-C) Representative images depicting the phase view merged with Cx43^GFP (green), ACE2^mCherry (red), and DAPI (blue, 10 µM and 10 min of exposure) labeling by HeLa-Cx43^GFP/ACE2^mCherry cells under control conditions (B) or treated with 50 pM spike S1 for 24 h (C). (D-E) Cx43^GFP and ACE2^mCherry labeling by Cx43^GFP/ACE2^mCherry cells showed in B and C. (F-G) DAPI staining by Cx43^GFP/ACE2^mCherry cells shown in B and C. (H) Averaged DAPI uptake rate normalized with the control condition (dashed line) by HeLa-parental, HeLa-Cx43^GFP, HeLa-Cx43^GFP/ACE2^mCherry or HeLa-ACE2^mCherry cells treated with 50 pM spike S1 for 24 h. *p < 0.05, **p < 0.01; effect of spike S1 compared to control (one-way ANOVA followed by Tukey’s post-hoc test). (I) Time-lapse measurements of DAPI uptake by HeLa-Cx43^GFP/ACE2^mCherry cells under control conditions (white circles) or treated with 50 pM spike S1 for 24 h (red circles). (J) Averaged DAPI uptake rate normalized to the maximum effect evoked by spike S1 (dashed line) by HeLa-Cx43^GFP cells acutely stimulated with different concentrations of spike S1. (J) Averaged DAPI uptake rate normalized with the control condition (dashed line) by HeLa-Cx43^GFP/ACE2^mCherry cells treated with 50 pM spike S1 for 24 h plus gap19 (100 µM), TAT-L2 (200 µM) or ¹⁰panx1 (100 µM). ***p < 0.001; effect of spike S1 vs. blockers (one-way ANOVA followed by Tukey’s post-hoc test). Data were obtained from at least three independent experiments with three or more repeats each (≥ 20 cells analyzed for each repeat). Calibration bar = 8 μm