Lipofectin

The two production clones CHO-K1/PG9 and CHO-K1/CN54gp140 were generated from a serum-free adapted host cell line derived from CHO-K1 (ATCC CCL-61). Transfection was performed using Lipofectin (Life Technologies). Briefly, 5 μg of PI-SceI (New England Biolabs) linearized BAC DNA was complexed with 25 μl Lipofectin in CD-DG44 medium (Life Technologies) containing 8 mM L-glutamine and 0.18% Pluronic F68 in a total volume of 200 μl. One million host cells, grown in CD-CHO medium (Life Technologies) supplemented with 8 mM L-glutamine were harvested in exponential growth phase and resuspended in 1.8 ml CD-DG44 medium with supplements. DNA complexes were added drop-wise to the cells and the transfection mixture was incubated in a humidified CO₂ shaking incubator (ISF1-X; Kuhner) for 24 hours at 37°C, 5% CO₂ and 125 revolutions per minute (rpm). 18 ml CD-CHO medium supplemented with 8 mM L-glutamine and 500 μg/ml G418 was added to the transfection mix and the culture was seeded in 96-well culture plates (Nunc, Thermo Fisher Scientific) at a volume of 100 μl per well. Plates were incubated in a humidified incubator at 37°C and 5% CO₂ until growth was observed (14–21 days). Positive wells were analyzed by ELISA. Clones from wells showing a high product concentration were subsequently expanded to a volume of 10 ml in T-25 cell culture flasks (Greiner Bio-One). The clones were monitored in routine culture for 3 passages before the best clone in terms of specific productivity and growth was used for single-cell dilution sub-cloning. Sub-cloning medium was prepared using 50% of 0.22 μm sterile filtered culture supernatant and 50% fresh CD-CHO medium supplemented with 8 mM L-glutamine and 500 μg/ml G418. Cells were diluted in sub-cloning medium and one cell per well was seeded in a 384-well cell culture plate (Corning) in a volume of 50 μl per well. Plates were incubated in a humidified incubator at 37°C and 5% CO₂. Grown wells were expanded to 96-well culture plates (Nunc, Thermo Fisher Scientific). The culture supernatant of single-clones was evaluated according to the product concentration and best performing clones were further expanded, banked and analyzed.

Replication assays were carried out as previously described [10 (link)]. Briefly, PHFG cells or SVG-A cells (2 × 10⁶ cells) grown in 75 cm² flasks were transfected either with JCV Mad-1 WT or JCV Mad-1 Pt mutant viral genome (8 μg/2 × 10⁶ cells/75 cm² flask) by lipofectin-2000 transfection method. Of note, The pBluescript (back bone) vector from both JCV and SV40 plasmids was digested with BamH I before using the plasmids in transfections. Lipofectin-DNA mixture was incubated with cells for 5 h and washed with PBS. Transfected SVG-A cells were fed with complete D-MEM media with 3% FBS. Transfected PHFG cells were fed with a special growth media D-MEM+F12 media containing 10% FBS, L-Glutamine (2 mM), gentamycin (50 mg/l) and insulin (2.5 mg/l). At indicated time points posttransfection, low-molecular-weight DNA containing both input and replicated viral DNA was isolated by the Hirt method [41 (link)], digested with BamH I and Dpn I enzymes, resolved on a 1% agarose gel and analyzed by Southern blotting.

Transient transfection for the purpose of Sirt-1 knockdown was implemented by antisense oligonucleotides (ASO) whose sequence was 5′-GTATTCCACATGAAACAGACA-3′. The sense oligonucleotides (SO) with a 5′-TGTCTGTTTCATGTGGAATAC-3′ sequence functioned as a control. Both substances were purchased from Eurofins (Ebersberg, Germany). During the experiments, the CRC cells were transfected by a suspension of 0.5µM Sirt-1-ASO or Sirt-1-SO in 10µl Invitrogen Lipofectin Transfection Reagent (Fisher Scientific, Schwerte, Germany) per ml cell culture medium containing 3% FBS. This method has been used previously (22 (link)).

Anti-PP2AC antibody (2038S), anti-phospho-LIMK1-Thr⁵⁰⁸/LIMK2-Thr⁵⁰⁵ (3841), and anti-phospho-AMPK-Thr¹⁷² (2531) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-PP2AC antibody (05-421) and metformin (1396309) was purchased from Millipore Sigma (Burlington, MA). Anti-actin (SC-477778) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). GFP-PP2AC cDNA (cat. #RG201334) and mouse monoclonal turboGFP antibody, clone OTI2H8 (TA150041) was from OriGene (Rockville, MD). PP2A phosphatase assay kit was purchased from Upstate. Lipofectin-RNAiMAX transfection reagent (13778030), Protein G Dynabeads (1003D), Alexa Fluor 488 Phalloidin (A12379), Prolong Gold Antifade (P36941), and SYBR Green PCR master mix (4309195) were obtained from Invitrogen (Waltham, MA). FuGENE HD transfection reagent (E2311) was from Promega (Madison, WI). The enhanced chemiluminescence Western blotting detection reagents (RPN2106) were obtained from Thermo Fisher Scientific (Waltham, MA). Human α-Thrombin (HT 1002a) from Enzyme Research Laboratories (South Bend, IN). ECIS electrodes (8W10E + PET) were procured from Applied Biophysics (Troy, NY).