Luminol

The second leaf from the top of 4- to 6-week-old rice plants was used for the measurement of ROS. Small leaf discs (approximately 4 mm in diameter) were cut from the leaves with a cork borer and pre-incubated overnight in sterile-distilled water. After the leaf disks were treated with elicitors, ROS generation was monitored by the luminol chemi-luminescence assay [26 (link)]. Three pre-incubated leaf disks per sample were immersed in a microcentrifuge tube containing 100 μl of luminol (Bio-Rad), 1 μl of horseradish peroxidase (Jackson ImmunoResearch), and the elicitor (100 nM flg22, 8 nM hexa-N-acetylchitohexaose, or water control). Luminescence was measured at 10-s intervals for 21 min using a Glomax 20/20 luminometer (Promega). Each treatment was represented by three replicate microcentrifuge tubes.

Protein samples of NHDFs treated with essential oils were extracted using Pro‐Prep solution (iNtRON Biotechnology, Seoul, Korea), according to the manufacturer's protocol. A bicinchoninic acid assay was used to determine protein concentrations. Proteins were then loaded and separated by 8% SDS/PAGE and transferred to nitrocellulose membranes using a wet transfer system. Membranes were blocked for 2 h with 5% skimmed milk in PBS containing 0.05% Tween‐20 (PBST) at room temperature. Subsequently, membranes were incubated with antibodies against collagen 1 (1 : 500; cat. no. sc‐293 182; Santa Cruz Biotechnology, Dellas, TX, USA), collagen 3 (1 : 500; cat. no. sc‐271 249; Santa Cruz Biotechnology), elastin (1 : 1000; cat. no. ab21736; Abcam, Cambridge, UK), and GAPDH (1 : 3000; cat. no. #2118; Cell Signaling Technology, Danvers, MA, USA), which served as the internal control, overnight at 4 °C. Blots were then incubated for 1 h with horseradish peroxidase‐conjugated secondary antibodies (1 : 5000; cat. nos. ADI‐SAB‐100 and ADI‐SAB‐300; Enzo Life Science, Farmingdale, NY, USA) in 5% skimmed milk in PBST for 1 h at room temperature. Luminol (Bio‐Rad, Hercules, CA, USA) was used to visualize antibody binding. Blots were scanned using Gel Doc 1000 version 1.5 (Bio‐Rad), and protein band intensities were normalized versus GAPDH.

For the preparation of whole cell lysates, samples were collected and lysed in lysis buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 150 mM KCl, 1 mM MgCl₂, 1 mM DTT, 5% Triton X-100, 10% glycerol, 0.1% NP-40, 1% Protease Inhibitor Cocktail, phenylmethylsulfonyl fluoride and 0.5% sodium dodecyl sulfate (SDS) (all from Sigma Aldrich, St. Louis, MO, United States). Protein concentrations were measured with the BCA Protein Assay Kit (Thermo Scientific, Rockford, United States). Samples (50 μg) were separated on a 7.5% SDS polyacrylamide-gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad, Hercules, United States), blocked in Tris-buffered saline (TBS) containing 5% Blotting-Grade Blocker (Bio-Rad, Hercules, United States). Membranes were incubated over-night at 4°C with primary anti-TNIP1 antibody diluted 1:500 and anti-actin (Sigma Aldrich, St. Louis, MO, United States) diluted 1:1,000. Subsequently, membranes were incubated for 2 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Texas, United States) secondary antibody diluted 1:2,000. Proteins were visualized with luminol (Bio-Rad, Hercules, United States) using a Omega Lum™ G Imaging System (Gel Company, CA, United States).

Cell lysates were prepared by using a mammalian cell lysis kit (Sigma, USA). The protein expression by western blotting was repeated thrice for each cell line. The following primary antibodies; polyclonal VEGFA, bFGF, TGFβ1 and E-cadherin (Abcam, UK), beta Actin (Bioss, USA) and EGFR (Santa Cruz, USA) were used. Following the peroxidase-conjugated secondary IgG (H + L) (Jackson Immuno Research, USA), immune complexes were visualized using luminol (Bio-Rad, USA). The chemiluminescence from the specific bands was visualized by Molecular Imager GelDoc XR+ ChemiDoc XRS+ Imaging System (BioRad, USA). The expression ratios of VEGFA, E-cadherin, TGFβ1, EGFR, bFGF, and β-actin from the blots were analyzed using Molecular Imager GelDoc XR+ ChemiDoc XRS+ Imaging System and Image Lab Software 6.0.1 (Bio-Rad, USA). After determining the intensities of the obtained bands, normalized protein fold ratios were calculated by proportioning the intensity value of each band to the corresponding β-actin band for each dose level.