6 cm cell culture dishes

HeLa (Expacy. Cellosaurus HeLa (CVCL_0030), n.d.) and human immortalized keratinocyte cell line HaCaT (Expacy. Cellosaurus HaCaT (CVCL_0038), n.d.) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Corning, NY, USA) containing 4.5 g/L of glucose, 10% (v:v) of fetal bovine serum (FBS) and 1% (v:v) of penicillin-streptomycin solution (100 U/mL penicillin and 100 μg/mL streptomycin). Cells were grown on 6 cm cell culture dishes (Corning) at 37°C in a humid atmosphere that contained 5% of CO₂ and were split every other day using trypsin-EDTA solution (Corning). For washing cells, Dulbecco’s Phosphate-Buffered Saline (DPBS) without calcium and magnesium was used. For experiments, cells were plated on 96-well plates (Thermo Fisher Scientific) at 1 × 10⁴ cells per well.
Pooled primary human epidermal keratinocytes (Promocell, Heidelberg, Germany) were cultured as previously described (Hermann et al., 2017 (link)). For the experiments, 4 × 10⁴ and 1 × 10⁴ cells were seeded in 24-well plates and 96-well plates, respectively, using a keratinocyte serum-free medium (KC-SFM) supplemented with recombinant epidermal growth factor (EGF), bovine pituitary extract (Gibco, Waltham, MA, USA), and antibiotic solution (5 U/mL penicillin and 5 μg/mL streptomycin).

Cell cycle analysis was performed as described previously [32 (link)]. To determine the cell cycle distribution, 5.5 × 10⁵ cells were seeded in 6 cm cell culture dishes (Corning) and allowed to adhere for 24 h at 37 °C and 5% CO₂. Afterwards, cells were treated with 50 µM of the respective compound. After 48 h, floating and adherent cells were harvested by trypsinization. Then, cells were centrifuged, washed in PBS, and fixed in 70% ice cold ethanol overnight at 4 °C. The DNA was stained using a staining solution containing 150 µg/mL PI and 0.5 mg/mL RNase for 30 min at rt in the dark. The DNA content was measured by a FACSCanto II (BD Biosciences) and the cell cycle distribution was determined using the FlowJo software (v7.6.5, FlowJo LCC).

The cells were isolated and cultured by the enzymatic digestion method as previously described [35 (link)]. In brief, three Holstein cows (at the fifth lactational stage) anterior pituitary glands were diced into small pieces of less than 1 mm³, and incubated in Hanks’ balanced salt solution without calcium and magnesium (CMF–HBSS) containing 0.3% I type collagenase, 0.1% hyaluronidase and 0.1‰ DNase (Sigma, Shanghai, China) at 37 °C for 2 h. The dispersed cells were washed three times with HBSS and resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, 25.0 mM glucose, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) at a seeding density of 1 × 10³ cells/mL. Then, the cells were seeded into a 75 cm² culture flask (Corning, Tewksbury, MA, USA) and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. After 6 d in culture, the cells were treated with 0.5% trypsin (Sigma) and 0.02% EDTA in CMF–PBS and then seeded into 6-cm cell culture dishes (Corning). The anterior pituitary cells at the second passage were used to perform the experiment.