Extended Data Figure 11

a and b, Inhibition of syncytia formation in Calu-3 cells by the downregulation of TMEM16F. Calu-3 cells were first silenced for each of the indicated genes or treated with siNTl (nontargeting siRNA1 as negative control) and then, after 24 hr, transfected to express the S-protein. After additional 24 hr, cells were immunostained for Spike (green) and nuclei (blue). Representative images are in a (scale bar: 500 μm), quantification in b. Data (mean±SD; n=3) are plotted as percentage of syncytia (cell area ≥ 20000 μm²) normalized on the total number of cells and expressed as fold over mock (lipid-only)-treated cells. *P<0.05, **P<0.01, one-way ANOVA with Dunnett post-hoc correction.

c and d. Effect of individual siRNAs forming the Dharmacon siRNA Pool against TMEM16F (ANO6). The picture shows the efficiency of syncytia formation after transfection of Spike cDNA in Vero cells in which TMEM16F was downregulated using the 4-siRNA Pool (which was used in all the other experiments in this study) or the individual siRNAs forming this Pool (siDH_1-4). We found that the commercial siDH_2 siRNA does not match with the TMEM16F sequence. Data in panel d are mean±SD, plotted as percentage of syncytia (cell area ≥ 20000 μm²) normalized on the total number of cells and expressed as fold over mock (lipid-only)-treated cells. **P<0.01, one-way ANOVA with Dunnett post-hoc correction.

e and f, Effect of selected siRNAs on MERS-CoV and SARS-CoV-2-induced syncytia formation. Vero cells were first silenced for each of the indicated genes (ACE2 and TMEM16F) or treated with siNT1 (non-targeting siRNA1 as negative control) and then, after 24 hr, transfected to express the either MERS or SARS-CoV Spike protein. After additional 24 hr, cells were immunostained for nuclei (white). Representative images are in in the panels on the left side, quantification in the bar chart on the right side. Data (mean±SEM; n=3) are plotted as percentage of syncytia normalized on the total number of cells and expressed as fold over mock (lipid-only)-treated cells. *P<0.05, **P<0.01, one-way ANOVA with Bonferroni post-hoc correction.

g, TMEM16F overexpression induces S-mediated syncytia formation. HEK293/ACE2 cells were co-transfected with human TMEM16A or TMEM16F together with a Spike-expressing plasmid. After additional 24 hr, cells were immunostained for Spike (green), TMEM16F (red) and nuclei (blue). Selected images and quantification in Figs. 5I and 5m. Scale bar: 200 μm.