Pegfp plasmid

Hippocampal and cortical tissue were harvested from embryonic day 17–19 Sprague Dawley rats of either sex, and then gently chopped and digested in 0.5% trypsin for 13 min at 37°C. Dissociated cells were plated at a density of 0.4×10⁴/cm² in a 35-mm dish with poly-L-lysine-coated coverslips in Neurobasal medium containing 1% horse serum, 0.5 mM glutamine, 1% antibiotic and 2% B27, at 37°C under 5% CO₂. After 6 h, the medium was replaced with Neurobasal medium containing 0.5 mM glutamine, 1% antibiotic and 2% B27. Subsequently, the culture medium was replaced every 5 d. At 5 days in vitro (DIV 5), cytosine arabinoside was added at a final concentration of 2.5 µM. In most experiments, primary hippocampal neurons were used at DIV 10 unless indicated otherwise. For morphological assessment of degeneration in individual neurons, the cultures were transfected at DIV 8 with the pEGFP plasmid (Clontech, Mountain View, CA) using Lipofectamine 2000 (Life Technologies) and used at DIV 10.

HCT116 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and penicillin/streptomycin and maintained at 37 °C and 10% CO₂. Stably transfected HCT116 cells were generated by transfection with pCMV-HPV16-E7 wt (kindly provided by Karl Münger^{48 (link)}), and selection with G418/Geneticin (PAA Laboratories, Pasching, Austria) at a concentration of 0.5 mg/ml^{41 (link)}. Wild-type mouse keratinocytes were isolated from C57BI6 mouse embryos as described previously^{49 (link)}. Cells were grown on plates coated with collagen (Invitrogen, Darmstadt, Germany) and maintained at 10% CO₂ and 32 °C in DMEM/Ham’s F12 (3.5:1.1) (PAN Biotech, Aidenbach, Germany). Cells were treated with doxorubicin (0.2 μg/ml; Medac, Wedel, Germany) or Nutlin-3a (10 μM; Cayman Chemicals, Ann Arbor, MI, USA) for 24 h. For cell sorting of transiently transfected wild-type mouse keratinocytes, pEGFP plasmid (Clontech, Mountain View, CA, USA) was co-transfected with pCMV-HPV16-E7 wt plasmid at a 1:3 ratio using GeneJuice (Merck, Darmstadt, Germany). Fluorescence-activated cell sorting was carried out on a FACS Aria SORP instrument (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA).

To determine the subcellular localization of ChPAP, the expression plasmid pYES2-ChPAP-EGFP was constructed. The ChPAP full-length sequence with restriction sites was cloned using PCR with EGFP-specific forward GFP-F (5'-GGATCCATGTTGCACGCGATGGTGGAC-3') and reverse GFP-R (5'-GGTACCCCAACAGGCACCATGCTGCTTGC-3') primers. The PCR product was ligated into the pEGFP plasmid (Clontech) digested with the BamH1 and KpnI sites. The ChPAP-pEGFP fusion fragment was digested at the BamHI and NotI sites in the pEGFP plasmid to construct the empty pYES2 vector.
The pYES2-ChPAP-EGFP and pYES2-EGFP plasmids were transferred independently into yeast cells. The transgenic yeast cells were pre-cultured in liquid medium containing 1% yeast extract, 2% peptone, and 2% glucose at 30°C for 2days. Afterward, they were washed three times to remove the remaining glucose. The EGFP and ChPAP-EGFP plasmids in yeast cells were induced to express in liquid yeast (1% yeast extract, 2% peptone, and 2% galactose) medium at 30°C for 6h, and then, ChPAP-EGFP yeast cells were incubated at 30°C with 20-μm FM4-64 dye for 3h. The remaining dye was removed by washing three times with sterile H₂O before samples were observed. The fluorescence was detected using laser-scanning confocal imaging system (Olympus Fluoview, FV500). The EGFP and FM4-64 signals were excited at 488nm and 543nm, respectively.