Fludarabine inhibits type I interferon-induced expression of the SARS-CoV-2 receptor angiotensin-converting enzyme 2

摘要

Fludarabine inhibits IFN-α-induced ACE2 expression via STAT1. a Immunoblot analysis of ACE2 in A549 and HBE cells treated with IFN-α for 6, 12, and 24 h. b, c After treatment with IFN-α for 12 h, ACE2 protein expression in A549 and HBE cells was detected by immunofluorescence (b) and flow cytometry (c). Scale bars = 10 μm. d Real-time PCR analysis of Ace2 gene expression in A549 and HBE cells treated with IFN-α. e HBE cells were treated with CHX (50 μM) and IFN-α for the indicated times and harvested for immunoblot analysis. f, g HBE cells were transfected with NC or Stat1 siRNA for 48 h and were then treated with IFN-α for 12 h. Cells were harvested for immunoblot (f) and real-time PCR (g) analyses (n = 3). h, i HBE cells were transfected with mock or Stat1 overexpression plasmids for 24 h and were then treated with IFN-α for another 12 h. Cells were harvested for immunoblot (h) and real-time PCR (i) analyses (n = 3). j Dual luciferase assay to evaluate the potential regulation of Ace2 promoter activity by STAT1 in 293T cells after cotransfection with pGL3-Ace2-3-luci and Stat1 expression plasmids and the Renilla luciferase reporter plasmid or empty vector for 24 h (n = 5). k ChIP analysis of the binding of STAT1 to the Ace2 promoter at positions −1232 to −1032 in HEK293 cells under normal or IFN-α stimulation conditions (n = 3). l–o HBE cells were treated with or without fludarabine (1 μM) for 12 h and were then treated with or without IFN-α for another 12 h. Cells were harvested for immunoblot (l), real-time PCR (m), immunofluorescence (n) and flow cytometric (o) analyses. Scale bar = 10 μm. p HBE cells were transfected with NC or Stat1 siRNA for 48 h and were then treated with fludarabine (1 μM) for 12 h and with or without IFN-α for another 12 h. Cells were harvested for immunoblot analysis. The data shown are the mean ± SD values and are representative of three independent experiments. Student’s t test was used for statistical analysis (n = 3). The concentration of IFN-α used in all in vitro experiments was 100 ng/ml. *P < 0.05; **P < 0.01; ***P < 0.001