Four well chamber slide

In four-well chamber slides (Thermo Fisher Scientific Inc.), cells were plated at a density of 4 × 10⁴ cells per well. Following treatment, the cells were tested in accordance with the manufacturer’s instructions ApopTag Peroxidase in Situ Apoptosis Detection Kit (Merck Millipore, Darmstadt, Germany). In a nutshell, cells were fixed in 1% paraformaldehyde in PBS 7.4 pH for 24 h at 4 °C. After being washed twice with PBS, cells were treated with TdT enzyme for 1 h at 37 °C. The slides were washed three times in PBS before being incubated with anti-digoxigenin peroxidase conjugate for 30 min at room temperature (RT) in a humidified environment, followed by three rinses with PBS at RT. The slides were then treated with the peroxidase substrate, counterstained with hematoxylin, dried, and mounted in a medium. The images were obtained using a light microscope with a 20× magnifying objective (Olympus BX43 with a camera and CellSens Programme, Hamburg, Germany).

Cell-proliferative potential was monitored by bromodeoxyuridine (BrdU) staining. Both types of cells were seeded at 5 × 10³ cells/cm² in four-well chamber slides (Thermo Fisher Scientific). On days 6 and 14 post seeding, 10 μM BrdU (Merck KGaA) was added to the cultures, which were then incubated for 3 h at 37 °C prior to fixation. BrdU incorporation was determined with mouse monoclonal anti-BrdU primary antibody (diluted 1:200; Roche Diagnostics) and Alexa 488-conjugated goat polyclonal anti-mouse IgG secondary antibody (diluted 1:1000; Life Technologies, Carlsbad, CA, USA). BrdU-positive cells were counted in 10 random fields under a confocal laser-scanning microscope (LSM 700; Carl Zeiss).

Fresh blood collected from female New Zealand white rabbits (Covance Research Products, Denver, PA, USA) was used in this study. Peripheral blood mononuclear cells (PBMCs) including monocytes were isolated using OptiPrep (Axis‐Shield, Oslo, Norway) according to a modified manufacturer's protocol for rabbit blood. Twofold diluted rabbit's blood was layered on OptiPrep density solution. After density gradient centrifugation at 700 g for 20 min with nonaccelerator and nonbrake modes, isolated PBMCs were washed two times with phosphate‐buffered saline (PBS; GE Healthcare Life Sciences Hyclone Laboratories, Logan, UT, USA) by centrifugation at 250 g for 10 min. Cells were resuspended in RPMI‐1640 medium (Life Technologies, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences Hyclone Laboratories) and 1% penicillin/streptomycin (Life Technologies) at a concentration of 2 × 10⁶ cells·mL⁻¹. Subsequently, cell suspensions were transferred to T75 tissue culture flasks (Sarstedt, Newton, NC, USA), four‐well chamber slides (Thermo Fisher Scientific, Rochester, NY, USA), or 24‐well flat bottom tissue culture plates (Becton Dickinson, Flanklin Lakes, NJ, USA). After incubation for 2 h at 37 °C in humidified 5% CO₂ in a gas incubator (Galaxy 170R; Eppendorf, Enfield, CT, USA), nonadherent cells were removed by washing with PBS 15.

Cells were seeded overnight in either 60 mm culture dishes or in four well chamber slides (Thermo Scientific). Lipofectamine RNAiMAX (13778075, Life Technologies), along with two hundred pmoles of each of the MDM2, SFRP1, SGOL1 and TTK siRNA constructs (Integrated DNA Technologies) or 5 μL of silencer negative control siRNA #1 (50 μM) (AM4611, Life Technologies) were transfected for 48 hours. siRNA sequences are presented in Table 1.