6 well cell culture plate

Skin biopsies were washed in HBSS containing antibiotics to remove blood, fat was trimmed away, and the remaining tissue was rinsed in 70% ethanol before being incubated in 50U/ml dispase (Gibco) at 37 °C for 2 hours to release epidermis from dermis. Epidermal sheets were incubated in 0.05% Trypsin-EDTA (Invitrogen) for 15 min at 37 °C, after which a slurry of cells was apparent. Trypsin was neutralized by the addition of complete Medium 154 culture medium containing 1% HKGS, 0.1 mM Ca²⁺ and antibiotics (Invitrogen). Cells were passed through a 100 µm cell strainer (BD Biosciences), washed, suspended in fresh culture medium, and seeded into 6-well cell culture plates (BD Biosciences). Cells were left undisturbed for 6 days at 37 °C with 5% CO₂. Thereafter medium was replenished every 2–3 days. Cells were harvested shortly after reaching confluence and further processed for RNA extraction.

MARC-145 cells and PAMs were prepared in 6-well cell culture plates (BD, Falcon) 24 to 48 h before the start of the experiment. Following this, the cell monolayers were pretreated for 2 h before infection with growth medium containing DiNap at five different concentrations (0, 0.01, 0.02, 0.04, and 0.06 mM). After the pretreatment incubation, the cell monolayers were washed with growth medium, inoculated with VR2332 at a multiplicity of infection (MOI) of 0.01 and incubated for 1 h. After the final 1-h incubation, the viral inoculum was discarded, and the cell monolayers were replenished with growth medium containing the same concentrations of DiNap as a treatment and incubated for 4 more days under the same culture conditions. The cell culture supernatants were collected every 24 h, centrifuged, and stored at −80 °C until analysis. The progeny virus titers were measured using a microtitration infectivity assay [52 (link)]; the virus titration assay was described briefly in a previous study [53 (link)]. The virus titers were calculated based on the cytopathic effect (CPE) and are expressed as TCID₅₀/mL [54 ].

Next, 5 × 10⁵ MIA-PaCa-2 or MCF-7 cells were plated into 6-well cell culture plates (BD Biosciences, San Jose, CA, USA) and transfected with the various plasmids as described [37 (link)]. Cells were transfected the WT-GSK-3β and KD-GSK-3β DNAs as described [37 (link)] with Lipofectin (Invitrogen) as described by the manufacturer. The generation of MIA-PaCa-2 + pLXSN cells were previously described [49 (link)]. Further, 48 h after transfection, a selection medium (DMEM + 5% FBS or RPMI-1640 + 5% FBS + 2 mg/mL G418 (Geneticin) (Invitrogen) was added to isolate stably transfected cells. Cells were fed with a fresh selection medium every three days. Mock transfections were also performed and did not generate viable colonies in the presence of selection medium.

Porcine intramuscular preadipocyte (PIP) cells, which are derived from marbling muscle tissue of the musculus longissimus thoracis from female Duroc pig [18 (link)], were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, Paisley, Scotland, UK) supplemented with 10% fetal calf serum (FCS), 100 mg/mL penicillin, and 100 U/mL streptomycin as a growth medium. PIP cells were plated at a density of 2.5 × 10⁴ cm² in 6-well cell culture plates (BD Falcon, Tokyo, Japan) and incubated at 37 °C in a humidified atmosphere of 5% CO₂. The medium was changed every day.

Human keratinocytes, cultured on feeder cells to ~70% confluence, were disaggregated in trypsin/EDTA and cells (5 × 10⁵) were seeded into 6-well cell culture plates (Falcon) coated with rat-tail collagen type I (20 µg ml⁻¹ in PBS, BD Biosciences), and cultured for 24 h in KSFM medium^{35 (link)}. Cells were then infected with MISSION® lentiviral particles (Sigma-Aldrich, the sequences of the shRNAs can be found in Supplementary Table 5) at a MOI of 3 in the presence of 5 µg ml⁻¹ polybrene (Sigma-Aldrich)^{35 (link)}. Medium was replaced after 24 h and shRNA-expressing cells were selected for 72 h using puromycin (2 µg ml⁻¹, Sigma-Aldrich)^{35 (link)}.