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. 2022 Apr 25;13:2237. doi: 10.1038/s41467-022-29896-z

Fig. 5. GATA6 is a novel regulator of ACE2.

Fig. 5

a WT-SARS-CoV-2-infected control and GATA6-disrupted Calu-3 cells were fixed 5 and 24 h post-infection and stained with antisera against SARS-CoV-2 (green) and DAPI (blue). Scale bar, 20 mm. Representative images of three biologically independent experiments are shown. b Western blot for the detection of GATA6 and ACE2 levels was performed in WT, ACE2 knockout, GATA6 knockout, and complemented Calu-3 cells. Representative images of two biologically independent experiments are shown. c ACE2 and GATA6 mRNA levels relative to GAPDH amount were analyzed in WT, NT control, ACE2 and GATA6 knockout Calu-3 cells by real-time PCR. Data were analyzed from four biologically independent experiments by one-way ANOVA with two-sided Tukey’s multiple comparison test. Shown are means ± SD. **p < 0.001; ****p < 0.0001. d Real-time PCR quantification of WT-SARS-CoV-2 in infected WT, GATA6 knockout and GATA6 knockout complemented by GATA6 or ACE2 expression Calu-3 cells. Cells were infected using MOI = 0.002 for 48 h. Data were analyzed from three biologically independent experiments by one-way ANOVA with two-sided Tukey’s multiple comparison test. Shown are means ± SD. *p < 0.05; **p < 0.005. e Cell viability of control, GATA6 knockout and GATA6 knockout complemented by GATA6 or ACE2 cells infected with WT-SARS-CoV-2 at MOI of 0.002 for 48 h, and stained with crystal violet. One of three repetitions is shown. f Cells transfected with a control or siRNAs targeting GATA6 were analyzed by real-time PCR for GATA6 and ACE2 expression relative to GAPDH amount. Data were analyzed from three biologically independent experiments by one-way ANOVA with two-sided Tukey’s multiple comparison test. Shown are means ± SD. ****p < 0.0001. g ACE2 ATG codon upstream sequences from nucleotide −1 to −820 are shown. Sequences shown framed in bold are potential GATA-binding sites. h Electrophoretic Mobility Shift Assays (EMSA) of lysates from HEK293T cells overexpressing GATA6 with labeled oligonucleotides derived from the promoter region of ACE2 results in the formation of protein-DNA complexes, which was prevented by competitive unlabeled oligonucleotides. Representative images of two biologically independent experiments are shown. Source data are provided as a Source Data file.