Elastin

Total tissue and cell lysates were assayed by Western blot according to our previous research 10. The antibodies Nrf2, Histione, extracellular signal‐regulated kinase (ERK), phosphor‐ERK, Jun N‐terminal kinase (JNK), phosphor‐JNK, p38, phosphor‐p38, anti‐rabbit‐HRP, anti‐goat‐HRP and anti‐mouse‐HRP were from Cell Signaling Technology (Danvers, MA, USA), and β‐actin, c‐fos, phosphor‐c‐fos, c‐jun, phosphor‐c‐jun, MMP‐1, procollagen type I, TGF‐β1, elastin and DLD‐E3 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Each experiment was repeated at least three times.

The total proteins were extracted from fibroblasts and then quantified by BCA method (BCA protein assay kit, Beyotime, China). The total proteins from each group were loading in the SDS-PAGE gel and separated by electrophoresis. Then the proteins were transferred onto a PVDF membrane. The membrane was blocked in 5 g/L skim milk for 1 h and washed in TBS. The membrane was incubated in the appropriate monoclonal antibodies at 4°C overnight. After washing in TBS-Tween (TBST), the membrane was incubated with HRP-conjugated anti-IgG secondary antibody (dilution) at 37°C for 1 h. After washing in TBST, the target proteins were visualized by using an ECL detection kit. We used antibodies to the following: GPX1(1:1000, Abcam, UK), COL1A1(1:500, Santa Cruz, USA), COL3A1(1:500, Santa Cruz, USA), Elastin(1:500, Santa Cruz, USA), MMP-2(1:500, Santa Cruz, USA), MMP-9 (1:500, Santa Cruz, USA), TIMP-2(1:500, Santa Cruz, USA), TGF-β1 (1:1000, Abcam, UK), β-actin(1:1000, Abcam, UK) and GAPDH (1:1000, Santa Cruz, USA) were used as an internal reference control.

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.

Paraffin-embedded sections were deparaffinized by 2 changes of clarene and rehydrated through an alcohol gradient. A heated antigen retrieval with 10 mM citrate buffer pH 6.0 was performed. Samples were blocked with 10% goat serum (Sigma-Aldrich) in phosphate-buffered saline for 30 minutes at room temperature and incubated with the unconjugated primary antibodies (α–smooth muscle actin [SMA], 1:100, Sigma-Aldrich; Isolectin B4-Biotin 1:100, Life Technologies; Elastin, 1:100, Santa Cruz Biotechnology) overnight at 4°C. Fluorophore-conjugated (Alexa Fluor 488 and Alexa Fluor 546, 1:400, Life Technologies) or chromogen-conjugated (horseradish peroxidase, 1:1,000, R &D Systems) secondary antibodies were incubated on the sections for 1 hour at room temperature in the dark. Incubation with 3,3’diaminobenzidine substrate (Abcam) was used for detection of horseradish peroxidase–derived signal. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:1000, Sigma-Aldrich) for fluorescent staining, or with hematoxylin for immunohistochemical staining. Slides were mounted with Hardset mounting medium (Vectashield). Images were taken with a Zeiss Observer.Z1 fluorescent microscope. ImageJ software was used to quantify the SMA and Elastin expression in the tissue sections.

Western blotting analyses were performed after 48 h of treatment. Cells were lysed in Radio-Immunoprecipitation Assay (RIPA buffer 1×; Cell Signaling Technology) and intra-cellular protein concentration was determined through the Bradford method. Specifically, 30 μg of proteins for each sample were resolved on a 10% SDS–PAGE gel and transferred onto nitrocellulose membrane (GE, Amersham, UK). Then, the membrane was blocked by 5% non-fat milk in Tris-buffered saline and 0.05% Tween-20 (TTBS) for 30 min and primary antibodies against Elastin (Santa Cruz, Dallas, TX, USA), Col I (Elabscience, Houston, TX, USA) and Col IV (Abcam, Cambridge, UK) were diluted 1:500 and incubated overnight at 4 °C. Secondary antibodies horseradish peroxidase-conjugated donkey anti-mouse and goat anti-rabbit were diluted 1:5000 and incubated for 2 h at room temperature. Anti-β-Actin antibody used at 1:1000 dilution was used as the loading control. The signal was detected using the ECL system (Chemicon-Millipore, Milano, Italy) and the semi-quantitative analyses of protein expression were carried out with the ImageJ program.

Paraffin-embedded sections were deparaffinized by two changes of clerene and rehydrated through an alcohol gradient. A heated antigen retrieval with 10 mM citrate buffer pH 6.0 was performed. Samples were blocked with 10% goat serum (Sigma-Aldrich, St. Louis, Missouri, United States) in phosphate-buffered saline for 30 min at room temperature and incubated with the unconjugated primary antibodies (α-SMA, 1:100, Sigma-Aldrich; Isolectin B4-Biotin 1:100, Life Technologies, Carlsbad, California, United States; Elastin, 1:100, Santa Cruz Biotechnology) overnight at 4°C. Fluorophore-conjugated (Alexa Fluor 488 and Alexa Fluor 546, 1:400, Life Technologies) or chromogen-conjugated (HRP-, 1:1000, R&D System) secondary antibodies were incubated on the sections for 1 h at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:1000; Sigma-Aldrich). Slides were mounted with Hardset mounting medium (Vectashield). Images were taken with a Zeiss Observer.Z1 fluorescent microscope. ImageJ software was used to quantify the smooth muscle actin (SMA) and Elastin expression in the tissue sections.