E1483

Transfections were performed by the calcium phosphate transfection method according to the standard protocols. Briefly, cells were seeded (3–5 × 10⁵) onto six-well plates. Twenty-four hours later, at 60–70% confluency, cells were co-transfected as follows: a mixture containing 1 μg DNA of pAdEasy-PY4-SV40-Luciferase or RGC-luciferase plasmid (the RGC sequence was amplified from RGC-mazE AdEasy system pShuttle vector with 5′-ATATATGCTAGCCCTGCCTGGACTT-3′ forward and 5′-ATATATAGATCTGATGGCCAGGCAAGTCC-3′ reverse primers. Then the RGC sequence was cloned into pGL3 promoter plasmid with NheI and BglII, upstream to SV40 promoter), 0.1 μg of Renilla luciferase plasmid (to evaluate transfection efficiency) (Promega, Madison, WI.cat. no. E223 Promega, Madison, WI, cat. no.E223), and 12.4 μl CaCl₂ (2.5 M) in sterile DDW at a final volume of 100 μl was added (dropwise) to a solution containing 100 μl 2× HeBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na₂HPO₄, 12 mM dextrose (glucose), 50 mM HEPES pH 7.05). After 15–30 min of incubation at room temperature, the mixture was sprinkled over the plate of cells.
Luciferase levels were measured after 72 h using the luciferase assay system (E1483, Promega, USA) and normalized to Renila luciferase activity according to the manufacturer’s instructions. Luminescence was measured by a LUMIstar® Galaxy luminometer (BMG Labtech, Germany).

BJ and HaCaT reporter cells were seeded in white 96-well plates (PerkinElmer) at concentration of 2 × 10⁵ in 100 μL of Minimum Essential Media (Sigma-Aldrich) supplemented by 1% nonessential amino acids (NEAA), 1 mM sodium pyruvate, 10% FCS, streptomycin (100 μg/mL), and penicillin (100 IU/mL) for 24 h at 37°C and 5% CO₂. The media was replaced by Opti-MEM media (Gibco) containing 0.5% FBS and 1% NEAA and cells were treated by 40 μg/mL of ginger extract or 30 μM quercetin (Sigma-Aldrich) while the control wells contained media with corresponding concentration of the solvent 0.05% dimethyl sulfoxide (DMSO) as described by Bak et al. [17 (link)]. Following 10 h treatment, the cells were lysed by 20 μL of cell culture lysis buffer (E153A, Promega) and 100 μL of luciferase assay substrate (E1483, Promega) was added. Luminescence was measured by EnSpire multimode plate reader (PerkinElmer). The values from luminescence assays were normalized by values acquired from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays with the cells that were prepared simultaneously with the same treatment conditions as described by Yamazaki et al. [18 (link)]. All the assays were performed three times with five technical replicates for each treatment.

To evaluate ITGB1 promoter transcriptional activity, SALL2 KO HEK293 cells were transiently co-transfected with 1 μg of ITGB1 promoter, 0.125 μg of RSV-β-galactosidase (β-Gal), and 2 μg of FLAG_SALL2 (pCDNA.3 FLAG SALL2) or 1 μg of vector control per well. After 24 h, the transfected cells were lysed with reporter lysis 5X buffer (E4030; Promega, Madison, WI, United States) and centrifugated at 14000 × g to pellet cell debris. The supernatant was then assayed for luciferase and β-Gal activity using the manufacturer’s suggested protocols (Promega; E1483 and E4740, respectively). Luminescent reporter activity was measured using a Luminometer (Victor3; Perkin- Elmer). All transfections were normalized to β-Gal activity and performed in triplicate. Luciferase values were expressed as fold induction relative to the pGL3 vector control.