Antibiotic antimycotic

HeLa cells were ordered from ATCC and maintained in RPMI 1640 medium, and HAP1 cells were purchased from Horizon Discovery and maintained in Iscove’s Modified Dulbecco’s medium. The ID8 mouse ovarian cancer cells were kindly provided by Dr. Guang Peng’s laboratory (MD Anderson Cancer Center). HeLa GRB2 deficient cells and HAP1 GRB2 deficient cells were generated by using CRISPR/Cas9 system as described previously^{11 (link)}. ID8 GRB2 stably knocked-down cells were generated by using GRB2 shRNAs which were purchased from Dharmacon (RMM4431-200333332, RMM4431-200335970, RMM4431-200399380, RMM4431-200404712). Media were supplemented with 10% fetal bovine serum (Lonza) and 1% antibiotic/antimycotic (Lonza) except The ID8 cells were maintained in DMEM supplemented with 4% fetal bovine serum (Lonza), 5 μg/mL insulin, 1% penicillin/streptomycin, 5 μg/mL transferrin, and 5 ng/mL sodium selenite. Cells were incubated at 37 °C in a humidified incubator with 5% CO2. Mycoplasma testing of these cell lines has confirmed negative results.

D407 cells (immortalized human retinal pigment epithelial cells) were cultured in DMEM (Cellgro, Mediatech, Inc., Manassas, VA) with 3% FBS (Atlanta Biologicals, Norcross, GA) and 1% Antibiotic/Antimycotic (Cellgro, Mediatech, Inc., Manassas, VA) at 37°C and 5% CO₂. HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% Antibiotic/Antimycotic. HCT116 cells (human colorectal cancer cells) were cultured in McCoy's 5A medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and 1% Antibiotic/Antimycotic. Each nucleofection reaction was comprised of one million cells and 1μg of each plasmid DNA. Nucleofection was performed using the Amaxa Nucleofector (Lonza). D407, HeLa and HCT116 cells were induced for apoptosis using 1 μM staurosporine (STS, Sigma, St. Louis, MO) prepared in DMSO.
D407 cells were a gift from Dr. Aparna Lakkaraju, originating from Dr. R. Hunt at University of South Carolina. HCT116^{BAX-/-/BAK-/-} and HeLa wild type cells were a gift from Dr. Richard Youle [34 (link)]. All cells used in these studies were wild type for endogenous BAX and BAK proteins, unless otherwise stated.

HEK293T cells obtained from the American Type Culture Collection were maintained in Dulbecco's modified Eagle's high glucose medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Lonza) in a humidified incubator with 10% CO₂ as described previously30 (link)31 (link)32 (link). Stable HEK293T cells overexpressing GFP-tagged FGFR2 were produced as described previously30 (link). Control and Grb2 knockdown cells were produced as described previously20 (link). Mammalian cell transfection was performed in a six-well plate using Metafectin Pro (Biontx-USA) reagents according to the manufacturer's instructions. Cells were lysed in 50 mM HEPES (pH 7.5), 1% (v/v) Igepal-C630, 1 mg ml⁻¹ bacitracin, 1 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 10% (v/v) glycerol, 50 mM NaCl, 1 mM PMSF and Protease Inhibitor Cocktail Set III (Calbiochem).

Saos-2 cells were obtained from the ATCC (Manassas, VA, USA). Whole-leaf teas were purchased from a local tea shop and the same batch was used to ensure consistency. Ham's F12 medium and antibiotic/antimycotic were purchased from Lonza (Mississauga, ON, Canada), whereas fetal bovine serum (FBS), and trypsin-versene were purchased from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). All other chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada). Laboratory consumables were purchased from Sarstedt (Nümbrecht, Germany) and optical density (OD) measurements were determined using a BIO-TEK Synergy HT Multi-Detection Microplate Reader (Winooski, VT, USA).

RAJI (Burkitt's lymphoma), U-937 (non-Hodgkin lymphoma), JURKAT (ALL), K-562 (chronic myeloid leukemia, CML), HL-60 (promyelocytic leukemia) MEG-01 (CML in megakaryocytic blast crisis), PC-3 (prostate cancer) cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). SH-SY5Y (neuroblastoma) and MDA-MB-231 (breast adenocarcinoma) were obtained from the American tissue culture collection (Manassas, VA, USA). All cell lines were cultured in RPMI 1640 medium (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal calf serum (Lonza) and 1% antibiotic–antimycotic (Lonza). Peripheral blood mononuclear cells (PBMCs) from human healthy donors were isolated and cultured as previously described [19 (link)]. All experiments were performed on cells in the exponential growth phase. Iso-3 was purified from Aplysina aerophoba [17 (link)] and dissolved in DMSO. Epigallocatechin gallate (EGCG), DAC, Necrostatin-1, VP-16, PP242 and bafilomycin A₁ were purchased from Sigma-Aldrich (Bornem, Belgium). SAHA and Z-VAD-FKM were purchased from Cayman Bio-connect (Huissen, The Netherlands) and from Millipore (Merck, Brussels, Belgium), respectively. All drugs were dissolved in DMSO. Recombinant human TRAIL was purchased by Enzo Life Science (Antwerpen, Belgium).

hBM-MSCs were a gift from the tissue culture lab of the Egyptian company for production of vaccines, sera, and drugs (Vacsera, Giza, Egypt). MSCs were cultured in complete culture medium (45 ml DMEM low glucose without L-glutamine (Lonza, Switzerland), supplemented with 5 ml fetal bovine serum FBS (Biowest, France), 0.5 ml antibiotic antimycotic (Lonza, Switzerland), and 0.5 ml L-glutamine (Lonza, Switzerland)) under standard culture conditions (37°C and 5% CO2). Upon reaching 70–80% confluency, the cells were passaged with trypsin-EDTA (0.25%) (Lonza, Switzerland) and re-suspended in complete culture medium.

The human HEPA-RG hepatoma cell line (Thermo Fisher Scientific, Waltham, MA, USA) was cultured using a hepatocyte bullet kit medium added with 10% FBS, depleted of exosomes (Lonza Biowhittaker, Oslo, Norway), and with 1% of 100 mM Antibiotic-Antimycotic (penicillin 10,000 91 u/mL, streptomycin 10,000 u/mL, (Lonza Biowhittaker, Oslo, Norway). The LX2 Human Hepatic Stellate cell line (Millipore, Merck Life Science, Milan, Italy) was cultured using Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA), added with 10% of FBS depleted exosomes, 1% of 100 M Antibiotic-Antimycotic, 2.5% of HEPES 1M (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) (Gibco™, ThermoFisher Scientific, Waltham, MA, USA) and 1% of 100 mM Sodium Pyruvate (Gibco™, ThermoFisher Scientific, Waltham, MA, USA). For all treatments with exosomes, both cell lines were cultured on semi-confluent cell monolayers and were treated with exosomes derived from patients at a concentration of µg exosome proteins/µL of medium, specifically at 20 µg/µL for 24, 48, and 72 h.