Fig. 2

The mechanism of coffee blocking SARS-CoV-2 entry was identified. a The activity of ground coffee and b instant coffee in different concentrations to block SARS-CoV-2 spike-ACE2 interaction was measured by ELISA. c Co-immunoprecipitation using 293 T cells expressing SARS-CoV-2 spike-HA and ACE2 to test coffee can inhibit the binding of ACE2 and spike. After anti-HA pulldown, adding instant coffee (2, or 4 mg/ml), then ACE2 and spike antibodies were used to detect by immunoblotting. dThe blocking capability of ground coffee and e instant coffee against SARS-CoV-2 entry was analyzed by cell-based TMPRSS2 enzyme activity assay. f Western blot was used to verify 293 T-ACE2 cells overexpressing TMPRSS2. g The impact of instant coffee tested by Vpp (MOI = 0.1) on TMPRSS2 involvement in SARS-CoV-2 entry in 293 T-ACE2 and 293 T-ACE2-TMPRSS2 cells. h Through Western blot to demonstrate coffee impacts cleavage of SARS-CoV-2 S by TMPRSS2. 293 T cells were transfected SARS-CoV-2 spike-HA and TMPRSS2-FLAG on 293 T cells for 6 h., then added with instant coffee for 24 h. i RT-qPCR was used to detect the regulation of the mRNA expression of ACE2 and TMPRSS2 on Huh7 (upper) and HepG2 (down) cells on instant coffee with 0, 0.25, 0.5, 1 mg/ml after 24 h. j Western blot to analyze the protein of ACE2 and TMPRSS2 on Huh7 and HepG2 treated with different doses of instant coffee after 24 h. k The quantification plots were analyzed from Western blot results. l, m The inhibition ability of instant coffee in different concentrations against wild-type SARS-CoV-2 on Huh7 and HepG2 cells was tested by Vpp (MOI = 0.1). n Huh7 cells were treated with different concentrations of instant coffee for CTSL activity, CL inhibitor as positive control. Statistical analyses were used by the Student T-test in triplicate independent experiments. Statistical significance was concerned as *P < 0.05, **P < 0.01 or ***P < 0.001