Penicillin streptomycin solution

Methanol, ethanol, chloroform, Cholesterol, DMSO, Amicon (Ultra-15-Membrane, MWCO 30000 ​Da), Span 20, Span 60, and Span 80 have been provided from Merck, Germany. Hyaluronic acid (MW 158 ​KDa) was taken from Changzhou Institute of Matera Medica Co. LTD. Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin solution were provided from PAN Biotech (Aidenbach, Germany). Trypsin-EDTA, Trypan blue, RPMI-1640 medium, PBS and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) were bought from Gibco, USA. Dialysis membrane (MWCO 12000 ​Da), Nile red, and Coumarin 6 were obtained from Sigma, USA. Human breast cancer cell lines, MCF- 7 and MDA-MB-231, NIH-3T3, SkBr3, and 4T1 cell lines were obtained from Pasteur Cell Bank, Iran. Epirubicin was purchased from Sina Darou, Iran. Annexin V/propidium iodide (PI) assay (i.e., Dead cell apoptosis kit) was purchased from Roche, Germany. RNA extraction kit Nucleic acid extraction kit was received from Qiagen, United States. The cDNA synthesis was done with Revert AidTM First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania).

HEK293 and COS‐1 cell lines were grown in Dulbecco's modified Eagle medium (DMEM) 4.5 g/l glucose w/o glutamine (Pan‐Biotech) supplemented with 10% (v/v) of FBS (ATCC) and 1% (v/v) of Penicillin/Streptomycin solution (Pan‐Biotech) at a temperature of 37°C in the presence of 5% of CO₂. Cells were plated in 6‐well plates and transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol with 1 μg of plasmid DNA. Cells were incubated for 48 h to ensure protein expression.

Experiments were performed using immortalized human airway epithelial cells (16HBE14o⁻) which had been derived from normal bronchial epithelial cells from a healthy donor [73 (link),74 (link)]. Cells were cultured on 10 cm Cell⁺ dishes (Sarstedt, Numbrecht, Germany) in Eagle’s MEM (PAN-Biotech, Aidenbach, Germany) containing 10% FBS superior (Merck, Darmstadt, Germany), 29.8 mmol/L NaHCO₃ and 1% (w/v) penicillin/streptomycin solution (PAN-Biotech, Aidenbach, Germany) at 37 °C and gassed with 5% CO₂. The cell culture medium was changed every 3 days. Cells were exposed to CYN for different periods as soon as they had reached coverage of 80% of the cell culture plate surface. Cells had formed confluent monolayers (Figure 1) by the end of the experiments. All cell cultures were checked for Mycoplasma contaminations regularly.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin solution were purchased from PAN Biotech (Aidenbach, Germany). Trypsin (TrypLE^TM Express 1X, cat. no. 12605-010) was purchased from Gibco (Thermo Fisher Scientific Inc, Waltham, MA, USA), and phosphate-buffer solution (PBS) from Corning (New York, NY, USA).
Sertraline, paroxetine, chlorpromazine, 5-FU, gemcitabine, and Thiazolyl Blue Tetrazolium Bromide were obtained from Millipore Sigma (Merck KGaA, Darmstadt, Germany) and dimethyl sulfoxide (DMSO, cat. no. D4540) was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Imatinib mesylate (cat. no. 5906) was purchased from Tocris Bioscience (Bristol, UK).

The murine melanoma cell line B16.F10 was cultivated in Dulbecco's modified Eagle's medium (DMEM) with the addition of 10% (V/V) fetal bovine serum (both from Sigma‐Aldrich Chemie GmbH), 1% (V/V) penicillin/streptomycin solution and 1% (V/V) l‐glutamine solution (both from PAN Biotech GmbH, Aidenbach, Germany) as described previously 11. HEK293 cells were cultured in DMEM supplemented with 10% (V/V) fetal bovine serum, 1% (V/V) penicillin/streptomycin at 37 °C and 5% CO₂ (Sigma‐Aldrich Chemie GmbH).
Tests for the absence of mycoplasms were performed routinely every month. LysoPC treatment of B16.F10 cells was performed with LysoPC C18:0 or LysoPC C18:1, achieving a final concentration of 450 μm and 2% (m/V) BSA for an incubation time of 72 h.
To prepare lysates of the LysoPC‐treated and untreated B16.F10 cells, cells in sub‐confluent flasks were washed twice with ice‐cold PBS (PAN Biotech GmbH) and incubated with lysis‐buffer and 1 mL of cell extraction buffer (both from Life Technologies GmbH, Darmstadt, Germany) for 10 min. Afterwards, cells were manually detached from the cell culture flasks and the cytosolic fraction was isolated via centrifugation at 17 000 g for 15 min.

The lACL-derived ligamentocytes were harvested from five adult males, healthy 12-month-old ACLs of New Zealand Rabbits, obtained from the regional slaughterhouse. The explanted LACLs were split into 2 mm² pieces and were cultivated in T25 culture flasks in ligamentocyte growth medium (Dulbecco’s Modified Eagle’s Medium [DMEM]/Ham’s F12 medium [1:1, PAN-Biotech] supplemented with 10% fetal bovine serum [FBS, PAN-Biotech], 1% penicillin/streptomycin solution [PAN-Biotech], 25 μg/mL ascorbic acid [Sigma-Aldrich, Munich, Germany], 2.5 μg/mL amphotericin B [PAN-Biotech] and MEM amino acid solution [Sigma-Aldrich]). After 7–10 days, ligamentocytes emigrated and were expanded using 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA, PAN-Biotech) for the subsequent experiments.

GHK and GHK-Cu were purchased from McBiotec (Nanjing, China). The ratio of GHK to Cu is 2:1 according to manufacturer data. Copper (II) chloride, 99%, and copper (II) acetate monohydrate, ACS reagent, ≥98%, powder were purchased from Sigma-Aldrich (St. Louis, USA). Phosphate buffered saline (PBS) (pH 7.4, 10×) was obtained from Vivantis (Malaysia). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, random primers and SYBR safe DNA gel stain were supplied by Invitrogen, Life Technologies (USA). Trypsin and penicillin/streptomycin solution were obtained from PAN-Biotech GmbH (Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were acquired from MP Biomedicals (Illkirch, France). RNeasy Mini Kit and QuantiFast SYBR Green PCR kit were purchased from Qiagen (Germany). random primers and avian myeloblastosis virus reverse transcriptase were purchased from Promega (Madison, Wisconsin, USA). Human interleukin 1 alpha and interleukin 8 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (San Diego, CA, USA). Human Fos-related antigen 1 (FOSL1) and Heat Shock 70 kDa Protein 1A (HSPA1A) ELISA kits were purchased prom MyBioSource (San Diego, CA, USA). All chemicals were used as supplied.