Dulbecco s phosphate buffered saline pbs

The 2D cultured rDPC and 3D spheroids were treated with or without 10 μM of limonin or 10 μM of minoxidil (Sigma-Aldrich) as a positive control for 24 h or 0–6 h. In some cases, cells were treated with wortmannin (10 nM) or XAV939 (10 μM) for 2 h, followed by treatment with limonin (10 μM) for 24 h, as described above. The cells were washed with Dulbecco’s phosphate-buffered saline (PBS; Welgene, Daegu, Korea) and lysed using the PRO-PREP^TM protein extraction solution (iNtRON Biotechnology, Seoul, Korea) for 1 h at 4 °C. Proteins (20 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat dry milk for 90 min and incubated with primary antibodies (Table 1) for 18 h at 4 °C. Membranes were washed and incubated with horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature. The protein bands were visualized on an X-ray film (Agfa-Gevaert, Mortsel, Belgium) using a Westar Nova 2.0 ECL solution (Cyanagen, Bologna, Italy).

BDB was purchased from Matrix Scientific (Colombia, SC, USA). Dulbecco’s modified Eagle’s medium and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Dulbecco’s phosphate-buffered saline (PBS) was obtained from Welgene (Daegu, Korea). Earle’s balanced salt solution (EBSS), hydrocortisone, insulin, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and minoxidil were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PRO-PREP protein extraction solution was obtained from iNtRON Biotechnology (Seoul, Korea). NE-PER^TM nuclear and cytoplasmic extraction reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lab-Tek^® chamber slides were purchased from Nalgene Nunc International (Rochester, NY, USA). Vectastain mounting medium was purchased from Vector Laboratories (Burlingame, CA, USA). Williams medium E, l-glutamine, and penicillin/streptomycin solution (Pen/Strep) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Westar Nova 2.0 ECL solution was obtained from Cyanagen (Bologna, Italy). Polyvinylidene fluoride (PVDF) membranes were purchased from Bio-Rad (Hercules, CA, USA). X-ray film was purchased from Agfa-Gevaert (Mortsel, Belgium). The Cyto-ID autophagy detection kit was purchased from Enzo Life Sciences (Villeurbance, France).