Penicillin g

The fruit calyxes of P. alkekengi var. franchetii collected in Icheon-si, Kyeonggi-do, Korea were purchased from Chodamchae Co. (Gunpo-si, Kyeonggi-do, Korea), in February 2011 and identified by one of the authors, Prof. Insop Shim (Kyung Hee University, Seoul 130-701, Korea). A voucher specimen (No. EA325) has been deposited at the College of Pharmacy, Ewha Woman’s University. Mouse macrophage RAW 264.7 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). Dulbeco’s Modified Eagle’s Medium; fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA); penicillin G (GE Healthcare Life Sciences, Logan, UT, USA); streptomycin (GE Healthcare Life Sciences, Logan, UT, USA); RP-18 F 254s (Merck, Darmstadt, Germany) plates; Kieselgel 60 F 254 (silica gel, 0.25 mm layer thickness, Merck, Darmstadt, Germany); TRIzol reagent (Gibco-BRL Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA); Taq polymerase (TaKaRa Co., Kusatsu, Shiga, Japan); SYBR green master mix (Invitrogen, Carlsbad, CA, USA).

C2C12 cells (ATCC^® CRL-1772™, <20 passages) were maintained in polystyrene flasks in a 37°C, 5% CO₂ incubator and cultured in growth medium (GM [1:1 Dulbecco’s Modified Eagle Medium (DMEM):F12 medium (11320-074, Invitrogen), 10% fetal bovine serum (FBS, 10270-098, Invitrogen), 10 U/mL penicillin G and 10 μg/ml streptomycin (15140-122, Invitrogen). Cells were subcultured prior to reaching 60%–70% confluence. For all experiments, C2C12 cells were seeded at 30,000 cells/cm² in GM until confluency (D0), when the medium was switched in differentiation medium (DM) (1:1 DMEM:F12 with 2% horse serum (HS from PAA Laboratories), 10 U/mL penicillin G and 10 μg/ml streptomycin as previously described (Ren et al., 2008 (link)). For the kinetics analysis of gene expression followed by qPCR, DM was refreshed at day 3 (D3) and day 5 (D5). Soluble BMP-2 (sBMP-2) was added in the GM at 600 ng/ml and refreshed at each change of medium until D5 (Figure 1A). Their translocations to the nucleus were observed by immunofluorescence at D0 for osterix, and at D1 and D3 for myogenin and MyoD, respectively. In addition, the ability of bBMP-2 films to induce later osteogenesis was confirmed by following the gene expression of Osteocalcin and by verifying cell mineralization.

African green monkey kidney fibroblasts (CV-1) and the human primary glioblastoma cell line U87-MG were obtained from the American Type Culture Collection (ATCC). IMA2.1 cells were obtained from S. Schildknecht (University of Konstanz, Germany). These cells are immortalized murine cortical astrocytes that respond to certain stimuli similarly to primary astrocytes with upregulation of specific mRNA resulting in expression of proteins [37 (link)]. BV-2 cells were kindly provided by M. Karlstetter (University Hospital Cologne, Germany). This cell line was derived from raf/myc-immortalized murine neonatal microglia and is the most frequently used substitute for primary microglia [38 (link)–40 (link)]. The murine glioblastoma cell line GL261 was kindly provided by A. Pagenstecher (Department of Neuropathology, University Hospital of Marburg, Germany). The identity of GL261 cells was confirmed by DNA sequencing by the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).
All cell lines were cultured in DMEM supplemented with antibiotic-solutions (100 U/ml penicillin G, 100 U/ml streptomycin, PAA Laboratories, Pasching, Austria) and with 10% fetal bovine serum (FBS; PAA Laboratories). Cultures were maintained and incubated at 37°C with 95% humidity and 5% CO₂. Growth medium was changed every third day until confluence.