Basal medium eagle

Colonies of sand flies L. longipalpis and L. migonei (both originally from Brazil) were maintained in the insectary of the Department of Parasitology, Charles University in Prague, under standardized conditions (26°C, fed on 50% sucrose and photoperiod 14 h light/10 h dark) as described previously (Volf and Volfova, 2011 (link)). Culicoides sonorensis biting midges were sent to Charles University from The Pirbright Institute, UK and kept at the same conditions as the sand flies.
Promastigotes of Porcisia deanei (MCOE/BR/91/M13541; TCC 258) isolated from C. nycthemera in Acara region, Brazil, P. hertigi (MCOE/PA/80/C8; TCC 260) isolated from C. quichua in 1980 in central Panama and L. infantum (MHOM/TR/2000/OG-VL) were cultured in M199 medium (Sigma) containing 10% heat-inactivated foetal calf serum (FBS, Gibco) and 2% sterile human urine (provided by J.Sa.) supplemented with 1% BME vitamins (Basal Medium Eagle, Sigma) and 250 µl/mL amikacin (Amikin, Bristol-Myers Squibb).
BALB/c mice originated from AnLab. s.r.o. (Harlan Laboratories, USA, Nederland). Animals were housed in T3 breeding containers (Velaz) equipped with bedding (German Horse Span Pferde) and breeding material (Woodwool), receiving standard ST-1 feed (Velaz) and water ad libitum, with a 12 h light/12 h dark photoperiod, temperature 22–25°C and humidity 40–60%.

For expression of ¹⁵N-labeled proteins, cultures were grown to OD 0.7 in 2YT, pelleted by centrifugation and the cell pellet was washed with 1 L M9 buffer (3 g L⁻¹ KH₂PO₄, 12.8 g L⁻¹ Na₂HPO₄ × 7H₂O, 0.5 g L⁻¹ NaCl) and resuspended in a quarter of the initial volume of minimal medium containing 4 g L⁻¹ glucose [unlabeled or uniformly ¹³C-labeled (U–¹³C-6, 99%, Cambridge Isotope Laboratories CLM-1396-10)], 1 g L^{−1 15}NH₄Cl (¹⁵N, 99%, Cambridge Isotope Laboratories NLM-467-25), 10 mL L⁻¹ Basal Medium Eagle (Sigma), 2 mM MgSO₄, and 0.1 mM CaCl₂ in M9 buffer. After a 1 h regeneration period at 37 °C, overexpression was induced with 1 mM IPTG.^{45 (link)} Cells were harvested, lysed, cleaved and purified as above for the equivalent unlabeled variants.

DPT was prepared by Professor Goo Yoon according to previous reports [42 ]. RPMI1640 medium, phosphate-buffered saline, fetal bovine serum, penicillin/streptomycin, and trypsin were purchased from Hyclone (Logan, UT, USA). MEM medium was purchased from Corning (Corning, NY, USA). DMSO, MTT, and Basal Medium Eagle were purchased from Sigma Chemical Company (St. Louis, MO, USA). Gefitinib was purchased from Cayman Chemical (Ann Abor, MI, USA). Primary antibodies against cyclin B1, cdc2, p21, p27, β-actin, GRP78, CHOP, DR4, DR5, Bcl-XL, Mcl-1, Bid, Bad, cyto c, α-tubulin, COX4, apoptotic protease activating factor-1 (Apaf-1), and c-PARP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-EGFR (Tyr1068), EGFR, p-GSK-3β (Ser9), GSK-3β, p-ERK (Thr202/Tyr204), ERK, p-AKT (Ser473), and AKT were purchased from Cell Signaling Biotechnology (Beverly, MA, USA).

Murine embryonic mesenchymal cell line C₃H/10T1/2 was obtained from ATCC (Wesel, Germany). Cells were grown in Basal Medium Eagle (Sigma-Aldrich, Milwaukee, WI, USA) supplemented with heat-inactivated fetal bovine serum (Gibco, Waltham, MA, USA) to a final concentration of 10% and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 40 μg/mL gentamicin sulfate (Sigma-Aldrich, St. Louis, MO, USA), under conditions of 5% CO₂ content in the air and at 37 °C. Cells of 7–10 passages were used in the experiments. The cells were planted in 96-well plates (SPL life science, Pocheon, Korea) at a concentration of 1.5 × 10⁴ cells/cm², cultured for 24 h and then added to OCP and OCP-Sr at concentrations of 10.00, 3.33, 1.11, 0.37, 0.12, 0.04, and 0.01 mg/mL and were co-cultured for another 96 h. OCP and OCP-Sr samples were pre-sterilized with 75% ethanol according to the indicated method [48 (link)].