Penicillin streptomycin

OS cell lines (U2-OS (HTB-06), Saos-2 (HTB-85), MG-63 (CRL-1427)) were of human origin (ATCC, Manassas, VA, USA). U2-OS and MG-63 cells were cultured in Eagle’s Alpha-Minimal Essential medium (EMEM) supplemented with 2.5 mM (w/v) L-glutamine, 10% (v/v) FCS, and 1% (v/v) penicillin/streptomycin (Gibco). Saos-2 cells were cultured in McCoy 5A medium (Lonza, Switzerland) supplemented with 2.5 mM (w/v) L-glutamine, 10% (v/v) FCS, and 1% (v/v) penicillin/streptomycin. OS cell lines were cultivated under standardized conditions (5% CO₂, 37 °C, 98% humidity) and medium was exchanged twice per week. Human osteoblasts (hFOB1.19), provided by the Core Facility Alternative Biomodels & Preclinical Imaging of the Medical University Graz, were cultivated at 34 °C in a 1:1 (v/v) mixture of Ham’s F12 medium and Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2.5 mM (w/v) L-glutamine, 10% (v/v) FCS, 1% (v/v) penicillin/streptomycin, and 0.3 mg/mL G-418 (Geneticin, InvivoGen, Toulouse, France). All cell lines were split at 80–90% confluence and cell numbers were determined by CASY (OMNI Life Sciences, Bremen, Germany). The mycoplasma test was performed every three months.

The monocyte/macrophage cell line J774 A.1 was grown in Dulbecco’s modified Eagle medium (DMEM) and Glutamax medium (Gibco, Darmstadt, Germany) containing 10% fetal calf serum (FCS, Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, Munich, Germany). The endothelial cell line MyEND, showing typical endothelial properties, was grown in DMEM with 10% FCS and 1% penicillin/streptomycin, as recently described [19 (link)].
The following agents were used for cell treatments: Pam₂ CSK₄, Pam₃ CSK₄ (Pam3), MAb-mTLR2, and mouse IgG from invivoGen (Toulouse, France); PD98059, SB203580, and SP600125 from Calbiochem (Merk, Darmstadt, Germany); and Bay 11–7082 from Enzo Life Sciences (Lörrach, Germany). The macrophage-activating lipopeptide of 2 kDa (MALP-2) was synthesized and purified as described before [20 (link)]. Before stimulation, cells were washed once with phosphate-buffered saline (PBS, Sigma-Aldrich) and incubated for the indicated periods in FCS-free cell culture medium containing the different agents at the indicated concentrations. The stimulation of cells with eRNA/Pam₂ CSK₄, 18S rRNA/ Pam₂ CSK₄, 18S rRNA/Pam₃ CSK₄, and 18S rRNA/MALP-2 mixtures was performed after preincubating both agents in double-distilled water for 30 min at 37 °C.

HEK 293 Gα_q/11 KO cells were generously provided by Dr Asuka Inoue and were described previously (56 (link)). The HEK 293 Gα_q/11 KO cells were cultured in Dulbecco’s modified Eagle’s medium (Corning, Cat # 10-017-CV) with 10% fetal bovine serum (FBS) (Gemini, Cat # 900-108) and 1% penicillin/streptomycin (Sigma, Cat # P4333). HEK 293 Gβ₁γ₂ stable cells were described previously (35 (link)) and were supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5 mg/ml of G418 (Invivogen, Cat # ant-gn-5). OCM-3, 92.1, and OMM1.3 cells were provided by Dr Andrew Aplin and have been previously described (19 (link)). UM001 cells were also described previously and obtained from Dr Takami Sato (57 (link)). The Mel202 cell line was from Dr Bruce Ksander. The OCM-3, 92.1, Mel202, OMM1.3, and UM001 cells were cultured in RPMI 1640 with L-Glutamine and 25 mM Hepes (Corning, Cat # 10-041-CV) supplemented with 10% FBS. All cell culture plates were obtained from Costar, Fisher, or GenClone.

Human neuroblastoma SH-SY5Y cells with inducible ΔK280 Tau_RD-DsRed expression [7 (link)] were maintained in DMEM/F-12 supplemented with fetal bovine serum (FBS, 10%) (Thermo Scientific, Waltham, MA, USA), penicillin/streptomycin (100 U/mL), selection antibiotics hygromycin (100 μg/mL) and blasticidin (5 μg/mL) (InvivoGen, San Diego, CA, USA), and 1.5 g/l sodium bicarbonate. Cells were cultured at 37 °C in a 5% CO₂ atmosphere. The cell viability of ZN and VB compounds (concentration range: 0.1–100 μM) was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) colorimetric assay. The absorbance at 570 nm was recorded by a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).

SKOV3 and A549 cells were cultured in McCoy's 5A and Ham's F-12 supplemented with 10% FBS plus 100 μg ml⁻¹ penicillin–streptomycin (WelGENE, Korea), respectively. SKOV3-RFP cells were cultured in DMEM plus 3 μg ml⁻¹ of puromycin (InvivoGen, UK) supplemented with 10% FBS and 100 μg ml⁻¹ penicillin–streptomycin. SKOV3 and A549 cells were purchased from Korean Cell Line Bank (KCLB, Korea) and SKOV3-RFP cells from Cell biolabs (USA).

All cells were cultured in a humidified incubator at 37˚C with 5% CO₂. Adherent cells were passaged by washing with PBS and sufficient incubation with 0.25% Trypsin-EDTA (Corning). Cultures were tested for mycoplasma every 6 months using the Lonza MycoAlertTM Detection Kit #LT07-318.
HEK293FT cells were cultured in IMDM (Caisson Labs) supplemented with 10% fetal bovine serum (FBS), 6mM L-glutamine, 0.1M non-essential amino acids (NEAA) (Corning), 500μg/mL Geneticin (Caisson Labs), 1% Pen-Strep, and prophylactic plasmocin (InvivoGen).
MCF10A cells were cultured in 50:50 DMEM/F12 (Corning) supplemented with 5% horse serum (HS) (Gibco), 6mM L-glutamine (Corning), 0.5μg/mL hydrocortisone (Sigma), 20ng/mL hEGF (PeproTech), 10μg/mL insulin (Sigma), 100ng/mL cholera toxin (Sigma), 1% penicillin/streptomycin, and prophylactic plasmocin (InvivoGen).
HCT116 and HCT116 p53^-/- were cultured in McCoy’s 5A medium with 10% FBS, 1% Pen-Strep, and prophylactic plasmocin.
Expi293F cell line (GibcoTM Cat# A14527, RRID:CVCL_D615) were maintained in Expi293™ Expression Medium (Gibco™) at 37°C with 8% CO2 and ≥80% relative humidity and 125 rpm shaking.