Microlumat plus

To establish HaCaT (3TP-luc) stable cells, cells were seeded on six-well plates. Cells were allowed to adhere overnight and then transfected with the p3TP-luc (neo) expression plasmid using PEI reagent (Sigma Aldrich). Transfected cells were cultured for four weeks in the presence of G418 (500 µg/mL). Several single clones were isolated and measured luciferase activity. The clone showing response to TGF-β1 treatment was used for reporter assay. HaCaT (3TP-luc) stable cells were seeded at 2.5 × 10⁴ cells/well in 96-well plate and were allowed to adhere overnight. Cells were concomitantly treated with TGF-β1 (2 ng/mL) and indicated concentrations of ALK5 inhibitors in 0.2% FBS medium and incubated for 24 h at 37 °C in 5% CO₂. Cell lysates were prepared using Luciferase Assay System (Promega) according to the manufacturer’s instruction, and luminescence was measured by a luminometer, Micro Lumat Plus (Berthold, Germany).

The cells were plated in 24-well plates at a density of 1×10⁵ cells/well and incubated at 37 °C in a humidified 5% CO₂ incubator. After 24 h, the cells were transiently transfected with pstat3-Luc reporter vector in the presence of pCMV-Luc vector using a transfection reagent (Intron Biotechnology, Seoul, Korea). At 24 h post-transfection, the cells were treated with compounds for 24 h. A luciferase assay was performed using the dual luciferase reporter assay system (Promega, Madison, WI, USA). The luminescence signal was measured using a luminometer (MicroLumat Plus, Berthold Technologies, Dortmund, Germany).

Plasmid DNA was purified using the DNA-spin^TM Plasmid DNA Purification Kit (iNtRON, Seongnam, Republic of Korea). Briefly, RAW 264.7 cells were transiently transfected with the pCMV-Luc and pNF-κB-Luc reporter vector using iN-fect™ in vitro Transfection Reagent (iNtRON). After incubation for 24 h, cells were pretreated with ebractenoid F for 2 h and then treated with LPS (1 μg/mL) for 8 h in 24-well plates. Cells were then lysed using passive lysis buffer (Promega, Madison, WI, USA). Luciferase activity was determined using a Dual-Luciferase Reporter Assay System (Promega) and measured with a luminometer (MicroLumat Plus, Berthold Technologies, Dortmund, Germany).

After 36 hof incubation, co-transfected cells were harvested, washed twice, and resuspended in 20µl medium. Luminescence was measured with the MicroLumat Plus (Berthold Technologies, Bad Wildbad, Germany) using the Dual-Glo Luciferase Assay system (Promega), according to manufacturer’s instructions. All experiments were performed in triplicate.

HEK293 cells (Sigma Aldrich) and the stable cell line HEK-TLR2YFP were used in reporter gene studies as described [2 (link)]. In brief, cells were seeded into 96-well tissue-culture plates at a density of 5 x 10⁵ cells/well. Cells were transiently transfected 16 hours later with an ELAM.luc reporter construct with TransIT-LT1 transfection reagent (Mirus Bio). Plasmid pcDNA was used to assure equal amounts of transfected DNA. The following day cells were incubated for 6 h with bacterial cells or cell wall extracts as indicated. After incubation, cultured cells were lysed in passive lysis buffer (Promega) and reporter gene activity was measured using a plate reader luminometer (MicroLumat Plus; Berthold).

pMITF-Gluc and pTYR-Gluc reporter systems harboring the promoter region of MITF and TYR were provided by Amore Pacific R&D Center (Seoul, Korea). pGL3-FL was obtained from S. Oh (Inje University, Busan, Korea). At 40%–50% confluency, the cells were washed with DPBS, and the Gaussia luciferase reporter construct pMITF-Gluc, pTYR-Gluc, and the control firefly luciferase vector (pGL3-FL) were transfected using FuGENE^® HD Transfection Reagent (Promega, Madison, WI, USA). After a 24 h incubation with GD, the cell lysates were prepared and the Gaussia and firefly luciferase activity were determined by the Gaussia luciferase assay kit (New England BioLabs, Ipswich, MA, USA) and luciferase reporter assay system (Promega, Madison, WI, USA), respectively, according to the manufacturers’ protocols, using a luminometer (MicroLumat Plus, Berthold Technologies, Dortmund, Germany).

A series of reporter plasmids carrying the CIITA-pIII promoter region just upstream of the luciferase gene in pGL4-Basic (Promega) were generated by using PCR and site-directed mutagenesis. The nucleotide sequences of synthesized oligonucleotides that were used as primers are listed in S1 Table. CAL-1 cells (5 × 10⁵) were transfected with 2 μg of pGL4.10-based reporter plasmid, and 2 ng of pRL-CMV (Promega) using Neon transfection system set at #4. Determination of luciferase activity was performed as described previously by using a luminomator, Micro Lumat Plus (Berthold Technologies, Bad Wildbad, Germany) or 1420 Luminescence Counter ARVO Light (Perkin Elmer) [20 (link)]. Renilla luciferase activity driven by pRL-CMV was used as an internal control to normalize the transfection efficiency.