2.1. Cell culture
Vascular smooth muscle cells (VSMC C591) were purchased from Pasteur Institute and were cultured in DMEM F-12 enriched with fetal bovine serum (FBS) 10% and Pen-Strep 1% using humidified incubator (CO2, 5%; 37 °C).
2.2. Cell viability assay
The effect of palmitic acid on the cell viability was carried out by microculture tetrazolium (MTT) colorimetric assay. Initially, the cells were grown in 96-well plate and were treated with the different concentrations of palmitate (0, 0.1, 0.5, 1, and 5 mM). After 24 and 48 hours’ incubations, the cellular medium was removed and the cells were re-incubated for 2 hours in the presence of MTT solution (200 µl, 0.5 mg/ml). Then, the produced formazan crystals were dissolved in DMSO (150 µl for 4 hours) and light absorbance was measured at 570 nm using microplate reader.
2.3. Treatment
Palmitic acid was solved in ethanol (1%), and was added to cultured cells (Confluency, 70%; Dose, 0.5 mM) for 24 hours. Then, the treated cells were harvested to evaluate the gene and protein expression levels.
2.4. RNA extraction, cDNA synthesis and RT-qPCR techniques
Total RNA was isolated from VSMCs using GeneAll-Hybrid-R purification kit (Cat. No. 305 − 101, GeneAll Biotechnology, Seoul, Korea). The quality and quantity of extracted RNA were evaluated using electrophoresis and Nanodrop 2000, respectively. The cDNA was synthesized using Kit (Cat. No. BR631-050, BioFACT 2 Step 2X RT-PCR Pre-Mix (Taq), Seoul, Korea). Gene expression quantitative analysis was performed by Applied Biosystems 7500 (Foster City, CA, USA) using Power SYBR Green PCR Master Mix Kit (Cat. No. A190303, Amplicon Denmark). The HCK primers were designed by primer blast (5’-CTCTTTGTCCGTGCGAGACT-3’, 5’- CCGTCGTTCCCCTTCTTGTA − 3’). GAPDH as a housekeeping gene was used to normalize the HCK gene expression level (Forward Primer: 5’-CATGAGAAGTATGACAACAGCCT-3’, Reverse Primer: 5’- AGTCCTTCCACGATACCAAAGT-3’).
2.5. Western Blot Analysis
Total protein was isolated using RIPA buffer containing protease inhibitors. Total protein concentration was measured by micro-lowry Kit (TP0200-1KT, Sigma, USA). The protein fractions were separated on 10% SDS-polyacrylamide gel (samples (11 µl) were loaded on the gel, and were run with voltage 80 V) for stacking gel (30 minutes) followed by higher voltage (110 V) for separating gel (60 minutes)) and, then were transferred on a polyvinylidene difluoride (PVDF) membrane (IPVH00010, Merck Millipore, Darmstadt, Germany; 80 V, 60 minutes). After blocking the PVDF membrane using milk blocking solution (5% milk, 60 minutes, room temperature), it was incubated separately with primary antibodies of HCK (E-AB-10359, Elabscience, China), p-Hck (phospho Y522; ab192578, abcam, United Kingdom), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-32233, Santa Cruz Bio-technology, CA, USA) overnight at 4 °C. Then, it was followed by the incubation with horseradish peroxidase-conjugated secondary antibody (sc-516102, Santa Cruz Bio-technology, CA, USA; 60 minutes, room temperature). Finally, the protein bands were observed by enhanced chemi-luminescence (ECL) reagent (RPN2235, Amersham, Italy) and were analyzed by ImageJ software (version 1.51, NIH).
2.6. Oil Red O staining technique
The intracellular lipid droplets were evaluated using Oil Red O staining method. Briefly, the cells were washed in phosphate-buffered saline (PBS) (2 times) and were fixed in formalin (4%) at room temperature for 45 minutes. Then, the cells were washed with isopropanol (60%) for 3 minutes. After adding Oil Red solution (0.1%, Cat. No. 1320-06-5, Sigma-Aldrich, USA), the cells were incubated at room temperature for 30 minutes. Finally, the cells were rinsed with water (3 times) and were observed by model IX71 microscope.
2.7. Statistical Analysis
Graph Pad Prism statistical software (v 8.3.0.538, Graphpad, USA) were applied for the data analyses. The differences between groups were determined by using independent samples t-student and Mann–Whitney tests. The IC50 was calculated in the dose-response way. P value lower than 5% was considered significant.