Plus reagent

CLDE cells were obtained as previously reported [18 (link)] and maintained in Ca²⁺-free keratinocyte serum-free medium (K-SFM, 37010–022; Gibco/Life Technologies) supplemented with epidermal growth factor, bovine pituitary extract, and 1% penicillin/streptomycin (Gibco/life Technologies) at 37°C in a humidified atmosphere of 5% CO₂. For the differentiation assay, cells were cultured with 100 ng/ml Neurotrophin (NT)-4 or 1.5 mM Ca²⁺. For transfection of a PKP1 expression vector or short interfering (si)RNA against Pkp1, cells were seeded in 12-well plates at a density of 2 × 10⁵ cells/well in K-SFM, then transfected with the expression vector using Lipofectamine 3000 with Plus reagent (Life Technologies) according to the manufacturer’s protocol. Cells were transfected with siRNA against Pkp1 (ON-TARGET Plus L-049579-01; Dharmacon, Lafayette, CO, USA) or control siRNA (D-001810-10; Thermo Fisher Scientific) using Lipofectamine 3000 without Plus reagent. To activate the Wnt signaling pathway, 5 ng/ml Wnt3a (R&D Systems, Minneapolis, MN, USA) or 40 mM LiCl [19 (link)] was added to cells in the presence or absence of Ca²⁺.

The complementary DNA to TP53 isoform RNA (kindly provided by Dr. J.C. Bourdon, University of Dundee, Dundee, UK) was cloned into a pcDNA3-expressing vector. H1299 cells were seeded into 6-well plates 24 h prior to transfection in order to reach an appropriate confluence (70–80%). Plus Reagent (Life Technologies, Carlsbad, CA, USA) or Turbofect (Thermo Fisher Scientific, Waltham, MA, USA) and p53 isoform expression plasmids (500 ng) were diluted in 100 µL of Opti-MEM medium (Life Technologies) with a ratio 1:1 (µL of Plus Reagent: µg of transfected DNA). Lipofectamine LTX Reagent (3:1) (Life Technologies) was diluted in 100 µL of Opti-MEM medium. Formed complexes were then added to plated cells and proteins were harvested 24 h later. For knockdown experiments, non-specific siRNA control scrambled or p53 isoform-specific small interfering RNA (siRNA) duplexes were synthesized at Eurogentec. The siRNA sequences used were as follows: si(-) (non-specific); p53α siRNA 5′-GUGAGCGCUUCGAGAUGUU-3′; p53β siRNA 5′-GGACCAGACCAGCUUUCAA-3′; p53γ siRNA 5′-CCCUUCAGAUGCUACUUGA-3′; pan-p53 (targets all isoforms) 5′-GACUCCAGUGGUAAUCUAC-3′. Reverse transfection was performed using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were harvested 48 h after transfection.