Dmem cell culture medium

Commercial Chlorella Pyrenoidosa powder and Antarctic krill oil were obtained from Dalian Jianyang Biological Company (Dalian, China) and Liaoyu Group Co., Ltd. (Dalian, China), respectively. The neutral proteases (EINECS:253-457-5) and alkaline proteases (EINECS:232-752-2) were obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Ferrous chloride and ascorbic acid were obtained from Damao Chemical Reagent Factory (Tianjin, China). Sodium dodecyl sulfate (SDS), linoleic acid (stored at −20 °C), 1,1,3,3-tetraethoxypropane, trichloroacetic acid, Thiobarbituric acid (TBA), Nile Red, and Nile Blue were bought from Aladdin Industrial Co., Ltd. (Shanghai, China). Ammonium thiocyanate was bought from Sinopharm Group Reagent (Shanghai, China). Trypsin-EDTA, fetal bovine serum (FBS), and DMEM cell culture medium were bought from Invitrogen (Carlsbad, CA, USA). A superoxide dismutase (SOD) assay kit and reactive oxygen species (ROS) assay kit were bought from Nanjing Jiancheng Bioengineering Research Institute (Nanking, China).

Human epidermal keratinocyte HaCaT cells were obtained from the American Type Culture Collection (ATCC^®, Manassas, VA, USA) and cultured at 37 °C in an incubator supplied with 5% CO₂ in Dulbecco’s Modified Eagle’s medium (DMEM) cell culture medium (Invitrogen Co., Carlsbad, CA, USA) treated with FBS (10%), streptomycin (100 mg/mL), and penicillin (100 U/mL). The medium was replaced twice a week. All the cells were serum-starved for 24 h before treatment with tart cherry and PM₁₀ in DMEM without FBS. To determine the relevance of signaling molecules in the apoptotic cell death pathway induced by PM₁₀, HaCaT cells were pretreated with N-acetylcysteine (NAC, 10 μM), SB203580 (10 μM), PD98059 (10 μM), and Bay 11-7082 (10 μM) for the inhibition of ROS, p38 mitogen-activated protein kinase (MAPK), ERK, and NF-κB for 30 min prior exposure to PM₁₀, respectively.

The human retinal pigment epithelial cell line ARPE-19 and the mouse fibroblast L929 cell lines were obtained from the American Tissue Culture Collection (ATCC, VA, USA). After thawing, the ARPE-19 cells were grown in Dulbecco’s Modified Eagle Medium and Ham’s F12 Nutrient Mixture (DMEM/F12, Gibco®, Invitrogen, Paisley, UK), and the L929 cells were grown in DMEM cell culture medium (Gibco®, Invitrogen). Both cell culture media were supplemented with 10% foetal bovine serum (FBS; Gibco®) and 1% antibiotic and antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B; Gibco®). These cell cultures were maintained in 75 cm² flasks (Costar, Corning Inc., Corning, NY, USA) in standard culture conditions of 37 °C and 5% CO₂, changing the media every 2 days. The cells were harvested by trypsinization using 0.05% Trypsin-EDTA solution (Gibco®) at 80–90% culture confluence and further sub cultivated into culture flasks^{8 (link),9 (link)}.

Doxorubicin (DOX, 20,170,511, purity >98%) was gotten from Beijing Yi-He Biotech Co. Ltd. All single-stranded DNAs were purchased from Takara Bio Co. (Dalian, China), and the specific sequences were displayed in Table 1. Phospholipids, 1,2-dioleoy-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesteryl hemisuccinate (CHEMS) were purchased from Avanti Polar Lipids (Alabama, USA), and N-(carbonyl-methoxy-polyethylene-glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolame (DSPE-mPEG2000) from Lipoid (Steinhausen, Switzerland). Cholesterol and calcein were obtained from Sigma-Aldrich Ltd (Auckland, New Zealand). PCR primer, loading Buffer, and Golden View was obtained from Beijing Ding Guo Chang Sheng Biotechnology Co. Ltd. Gel Extraction Kit, 2× Taq Master Mix, and 50× TAE was obtained from Beijing Com Win Biotech Co. Ltd. Sulforhodamine B (SRB), DMEM cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS), and heparin sodium were bought from Gibco Invitrogen. DAPI, hematoxylin and eosin were supplied by Beyotime Biotechnology Co. Ltd. Other reagents were acquired from China National Medicine Corporation Ltd.

The SW982 cell line (ATCC, USA) was maintained in DMEM cell culture medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (HyClone, USA), 100 units/ml penicillin, and 100 units/ml streptomycin. A synthetic 3′-UTR of ATF2 with the putative miR-204/211 seed region was inserted into the multiple cloning site of the pmiR-RB-Report (Thermo Fisher, USA) vector. SW982 cells were cotransfected individually with wild-type 3′-UTR or the mutant sequence and miR-204 mimics (RiboBio, Guangzhou, China) or a small RNA negative control. We collected the lysates 36 hours after transfection and evaluated the activity of renilla luciferase according to the manual.

The immortalized embryonic mouse hypothalamus cell line N 25/2 (mHypoE-N25/2, Cellutions Biosystems Inc., Canada) was cultured in DMEM cell culture medium (Invitrogen) with 10% Fetal Bovine Serum (Invitrogen), 1% Penicillin Streptomycin (Invitrogen), 1× Amphotericin B (Invitrogen) in a humidified atmosphere with 5% CO₂ in air at 37°C. Growth medium was changed every 2 days and cells were split with trypsin regularly with a ratio of 1∶2–1∶3 usually every 3 days.