Cell cultivation and treatment
Human lung cancer A549 cells and human adenocarcinoma HT29 cells were cultivated in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma Aldrich, Czech Republic)) supplemented with 10% fetal calf serum (FCS) (Merck, Germany). A549 lung cancer cells were treated by PARP inhibitor, olaparib (final concentration 10 µM for 24 hours; #S1060, Selleckchem, Germany), an inhibitor of histone deacetylase, SAHA (final concentration 5 µM for 24 hours; #10009929, Cayman Chemical Company, USA). Also, A549 cells were treated by antimalarial chloroquine diphosphate (final concentration 16 µM for 24 hours, #S4157, Selleckchem, Germany), ACE inhibitor lisinopril (100 nM for 24 hours; #S2076, Selleckchem, Germany), vitamin D2 (100 nM for 24 hours; #S4035, Selleckchem, Germany), or by combination chloroquine diphosphate (16 µM for 24 hours), vitamin D2 (100 nM for 24 hours) and ACE inhibitor (100 nM for 24 hours).
The human intestine adenocarcinoma (HT29) cells were treated by the inhibitor of histone deacetylases (HDACs), sodium butyrate (NaBt; final concentration 5mM; #B5887, Sigma Aldrich, Czech Republic) inducing enterocytic differentiation for 48 hours treatment see Bartova et al. (2005). In these types of experiments, we studied the effect of histone hyperacetylation and cell differentiation on the level of the ACE2 protein.
We also study the ACE2 level in human embryonal kidney cells, HEK293, cultivated in 293SFM II (#11686029, Thermo Fisher Scientific, Czech Republic) growth medium. This is a serum-free, protein-free medium for growth and transfection of the suspension-adapted human embryonic kidney (HEK) 293 cells. For the cultivation, the medium was supplemented by 10% FCS (Merck, Germany).
For irradiation by cobalt–60, cells were cultivated on Petri dishes, irradiated by the dose of 5Gy of γ-rays delivered by irradiation device Chisostat (the Chirana, Czech Republic). Cells were either fixed by 4% formaldehyde for immunostaining experiments or harvested for western blots 2 hours after γ-irradiation.
Cultivation of mouse ESCs and differentiation into cardiomyocytes
Were studied following mESC lines: wild-type mESCs, D3 line (mESCs wt, wild-type), and mESCs that were characterized by histone deacetylase 1 (HDAC 1) deficiency (HDAC1 dn mESCs) (Zupkovitz et al. 2010). Mouse ESCs were cultivated on 0.2% gelatine-coated Petri dishes (valid for wt-cells) or Matrigel (#354277, Corning Incorporate, USA)-coated plastic dishes (valid for HDAC1dn-cells). Mouse ESCs were grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma Aldrich, Czech Republic).
The medium was supplemented with penicillin and streptomycin, 0.1 mM non-essential amino acids, 1 ng/ml mouse leukemia inhibitory factor (LIF), 100 μM mono-thioglycerol, and 15% fetal bovine serum (FBS). Differentiation into cardiomyocytes via the formation of embryonic bodies (EBs) was performed following Kudova et al. (2016) and Arcidiacono et al. (2018). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Differentiation into cardiomyocytes was activated by cell seeding into ES culture media without leukemia inhibitory factor (LIF). In this step, we used the “hanging drop” method. In day 3 of cultivation, EBs were placed on new plastic dishes; in day 6, EBs were transferred to gelatine-coated culture dishes with DMEM/F12 (1:1) (#11320–033, Sigma Aldrich, Czech Republic) supplemented with insulin, transferrin, and selenium (ITS–100x, #41400–045, ThermoFisher Scientific, Czech Republic) (DMEM/F12-ITS). The differentiation medium (DMEM/F12-ITS) was changed every two days. The duration of the differentiation into cardiomyocytes was up to day 20 (dd20).
Tissue isolated from experimental animals.
Explanted hearts from mouse strain C57Bl6 were studied on the distribution pattern of the ACE2 protein and its interacting partner, renin. Mice were kept in a pathogen-free (SPF) animal facility (see description in Arcidiacono et al. 2018). For experiments, we needed the agreement of the Ethics Commission of the Ministry of Agriculture of the Czech Republic (protocol No.48/2016). After breading, embryos were explanted from female animals 15 days post-conception (e15), and embryonic hearts were treated by HDAC inhibitors (200 nM TSA, 16 µM SAHA, and 15 mM VPA) (more detail see Vecera et al. 2018). Except for embryonic and young, old female and male hearts, we additionally isolated adult mouse lungs, brains, and kidneys. By the use of western blots, we compared the level of the ACE2 protein in selected tissues.
Cell culture immunostaining
Immunofluorescence was performed following Legartova et al. (2019). Briefly, the cells were fixed in 4% formaldehyde (PFA) for 10 min at room temperature (RT), permeabilized with 0.2% Triton X–100 (#194854, MP Biomedicals, USA) for 10 min and 0.1% saponin (#S7900, Sigma Aldrich, Czech Republic) for 12 min. After that, the dishes were washed twice in phosphate-saline buffer saline for 15 min. We used 1% bovine serum albumin (BSA; #A2153–506, Sigma Aldrich, Czech Republic) dissolved in 1x PBS as a blocking solution. Next, the samples were incubated in the blocking solution for one hour at room temperature and then washed in 1x PBS for 15 min. For immunofluorescence analysis, the following antibodies were used: anti-ACE2 (#ab15348, Abcam, UK), anti-renin (#PA5–102432, ThermoFisher Scientific, Czech Republic) and α-actinin (#A7811, Sigma Aldrich, Czech Republic). As secondary antibodies, we used: Alexa Fluor 594-conjugated goat anti-rabbit (#A11037, ThermoFisher Scientific, Czech Republic), Alexa Fluor 594-conjugated goat anti- mouse (#A11032, ThermoFisher Scientific, Czech Republic), Alexa Fluor 488- conjugated goat anti-rabbit (#ab150077, Abcam UK). Negative control was considered samples incubated without primary antibodies. Cell nuclei (GC-rich sequences of DNA) were stained by 4′,6-diamidino–2-phenylindole (DAPI; Merck, Czech Republic). As a mounting medium, we used Vectashield (#H–1000, Vector Laboratories, USA).
Heart tissue cryosectioning
Fixed mouse hearts were frozen in embedding medium (OCT embedding matrix, Leica Microsystems, Mannheim, Germany) at −80°C. Mice hearts were sectioned using a Leica Cryo-microtome (Leica CM 1800, Leica, Germany). The cryo-sections were washed in PBS. The thickness of the sections was 13–14 µm. For the immunofluorescence technique, we used the protocol published in Vecera et al. (2018). The primary antibody was anti-ACE2 (#ab15348, Abcam, UK), as the secondary antibody we used Alexa Fluor 594-conjugated goat anti-rabbit (#A11037, ThermoFisher Scientific, Czech Republic). DNA was counterstained using 4′,6- diamidino–2-phenylindole (DAPI; Merck, Czech Republic), and the mounting medium was Vectashield (Vector Laboratories, Vector Laboratories, USA).
Cryo-sections of whole embryonic hearts (at embryonal stage e15) were stained by appropriate antibodies, as mentioned above. For analysis, we used the “tile-scanning” mode, a toll of Leica SP–5 confocal microscope (Leica, Germany). To acquire images, we used HCX PL APO lambda blue 20.0× 0.7 IMM UV objective (Leica Microsystems, Germany) (see Bartova et al. 2016).
Laser scanning confocal microscopy
For analyses, we used a Leica TCS SP8-X SMD confocal microscope (Leica Microsystem, Germany), equipped with 63× oil objective (HCX PL APO, lambda blue) with a numerical aperture (NA) = 1.4. Image acquisition was performed using a white light laser (WLL; wavelengths of 470–670 nm in 1 nm increments) with the following parameters: 1024 × 1024-pixel resolution, 400Hz, bidirectional mode, and zoom 8–12 using the Leica Application Suite (LAS X) software.
Western blots were performed following Jugova et al. (2011). Cell cultures or tissue samples were washed with PBS and lysed in sodium dodecyl sulfate (SDS) lysis buffer (50 × 103 mol/l Tris-HCl, pH 7.5; 1% SDS; 10% glycerol). The total protein concentration was determined by the DC protein assay kit (#5000111, Bio-Rad, Czech Republic) and ELISA Reader μQuant (BioTek, USA). The proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 2% fat-free milk or 2% gelatin for one h and then immunoblotted overnight at 4°C with the following primary antibodies against the ACE2 protein (#ab15348, Abcam, UK). Primary antibodies were diluted 1:1000, and as a secondary antibody, we used goat anti-rabbit IgG (#AP307P, Merck, Czech Republic; 1:2000). The western blot data were normalized to the amount of total protein and were quantified by using ImageJ software (NIH freeware). The Student’s t-test was used for statistical analysis (Sigma Plot software version 14.0, Jandel Scientific, USA). Statistical significance at p ≤ 0.05 is shown by (*).
Statistical analyses and quantification of fluorescence intensity
For analysis of western blot fragment density and analysis of fluorescence intensity, we used ImageJ (NIH freeware) and ImageQuant TL software. For statistical analysis, we used the Student’s t-test. Experiments were repeated trice.