Cell culture with or without HDACIs. The cell lines KATOIII (JCRB0611), K562 (JCRB0019), NUGC-4 (JCRB0834) and MKN1 (JCRB0252) were originally purchased from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), SW480 (ATCC CCL-228), SV-HUC (ATCC CRL-9520), HMVEC-L (ATCC CC-2527) and SH-SY5Y (ATCC CRL-2266) were from American Type Culture Collection (Manassas, VA), 5637 (TKG 0605), KK47 (TKG 0663) and T24 (TKG 0443) were from Cell Resource Center for Biomedical Research (Miyagi, Japan) and LAN-5 (RCB0485) was from RIKEN BioResource Research Center (Ibaraki, Japan). The KATOIII and K562 cells were cultured as described previously . The NUGC-4, MKN1, SW480, 5637, KK47 and LAN-5 cell lines were cultured in RPMI1640 medium containing 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. The culture medium for SV-HUC, T24, HMVEC-L or SH-SY5Y was Ham’s F12K, MEM, EGM™-2MV Microvascular Endothelial Cell Growth Medium-2 or 1:1 mixture of MEM and F12, each with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin. For treatment with the HDACIs, the cells were seeded at a density of 2.5×105/ml one day before the experiment. On the following day, the cells were re-seeded at a density of 2.5×105/ml in new medium with or without HDACIs. The medium was not changed thereafter until harvest of the cells. The HDMCIs we used included sodium butyrate (#303410; Sigma-Akdrich), panobinostat (#13280; Cayman Chemical Company), sodium valproate (#13033; Cayman Chemical Company), vorinostat (#10009929; Cayman Chemical Company) and trichostatin A (#89730; Cayman Chemical Company). The solvent used for sodium butyrate and sodium valproate was deionized-distilled water, and that used for panobinostat, vorinostat and trichostatin A was dimethyl sulfoxide.
Quantitative real-time PCR (qPCR). RNA purification, cDNA preparation and quantification of ABO and ACTB transcripts were performed as described previously . qPCR of the ACE2 and TMPRSS2 transcripts was performed with the specific primer sets “ACE2 Primer 2” and “TMPRSS2 Primer 2”, respectively, as described by Ma et al. , under the following conditions: 95°C for 3 minutes and 40 cycles at 95°C for 3 seconds and at 60°C for 30 seconds. Every assay was conducted at least twice, and the absolute amount of each transcript determined by qPCR was standardized by the amount of ACTB transcript.
Enzyme-linked immunosorbent assay (ELISA). ELISA was performed using a Human ACE2 ELISA Kit (#ab235649, Abcom) following manufacturer’s instructions. Briefly, KAKTOIII or NUGC-4 cells were harvested 24 or 48 hours after incubation with or without HDACIs, and solubilized in 1× cell extraction buffer PTR. After centrifugation, the concentration of total protein in the supernatant was measured using a DC protein assay kit (#5000112JA, Bio-Rad), and 250 or 100 ng of total protein derived from KATOIII or NUGC-4 cell lysates, respectively, was applied to each well of a ready-to-use microplate provided in the kit. Then the ACE2 antibody cocktail was added to each well, followed by 1-hour incubation at RT on a plate shaker set to 400 rpm. After the incubation, each well was rinsed three times, TMB Development Solution was added, and incubation was performed at RT on a plate shaker. Ten minutes later, the Stop Solution was added to each well, and the end point reading of OD at 450 nm was recorded using a iMark™ microplate absorbance reader (#168-1135, Bio-Rad). The concentration of the ACE2 protein in the sample was determined by interpolating the blank control subtracted absorbance values against the standard curve. Every assay was conducted in duplicate.