Flp in 293 cells

Flp-In 293 cells (catalog number R750-07), pCDNA5/FRT/TO, and pOG44 were obtained from Life Technologies. Flp-In 293 cells were maintained in DMEM high-glucose w/L-glutamine supplemented with an additional 2 mM L-glutamine (Gibco) and 100 µg/mL zeocin (Life Technologies). Haps59 and yCD were cloned into pCDNA5/FRT/TO, and DNA for transfections was prepped from overnight cultures of E. coli using PureYield Miniprep kit (Promega). Α total of 2.7 µg of pOG44 and 300 ng of pCDNA5/FRT/TO-EV, -yCD, and -Haps59 were transfected into 100,000 Flp-In 293 cells, plated eighteen hours prior in a single well of a 6-well plate using the CalPhos Mammalian Transfection Kit (Clontech) following the manufacturers directions. Twelve hours after transfection the cells were washed with PBS and fresh media was added. Thirty-six hours after transfection the cells were split 1 to 5 into a fresh 6-well plate of media containing 150 µg/mL hygromycin-B, instead of zeocin. Media was changed every 3–4 days until foci formed, in about 2 weeks. These stable pools were passaged 3 times, at a 1/10 dilution into 200 µg/mL hygromycin-B, before being used in toxicity assays.

Cre proteins were expressed using E. coli BL21(DE3) strain carrying the plasmid pETHNCre. E. coli was cultured at 25 °C for 6 h following 0.1 mM IPTG induction. Cre proteins were purified using Ni-NTA column with 20 mM imidazole wash and 500 mM imidazole elution buffer containing TBSG (10 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 8.0). Proteins were dialyzed against HBSG (20 mM HEPES, 150 mM NaCl, 10% glycerol, pH 7.4). A hydrophobized nanoneedle array was immersed with 10 μl of 100 μM Cre protein solution and incubated at RT for 60 min. The array chip was then rinsed with HBS buffer before fixing on the array holder. We prepared 293.RxG cells by the following method: A plasmid pcDNAFRTxE2CxmEm, which encodes GFP (mEmerald) and RFP (E2-Crimson) sandwiched by two loxP sites, was stably integrated into Flp-In-293 cells (Life Technologies) according to the manufacturer’s protocol. A clone of 293.RxG was selected with hygromycin, which expresses RFP at normal conditions. The method for culturing 293.RxG cells and insertion of the nanoneedles into cells, were same as described in the previous section. We observed fluorescence change 48 h after treatment. We calculated the efficiency of delivery from the area of GFP-expressing cells and RFP-expressing cells using Image-Pro Plus.

Flp-In 293 cells (Life Technologies) stably expressing FLAG-tagged human LRRK2 or parent cells were grown in suspension culture (Joklik-modified Eagle’s minimum essential medium with 5% (vol/vol) fetal bovine serum [FBS]). The cell pellet (2.4 x 10⁸ cells) was homogenized in lysis buffer (50 mM Tris pH 7.4, 120 mM NaCl, 5 mM EDTA, 10% (vol/vol) glycerol, 1% (vol/vol) Trion-X100) supplemented with Complete protease inhibitor cocktail (Roche Applied Science). The soluble fraction of the suspension was immunoprecipitated with anti-FLAG M2 affinity gel (Sigma-Aldrich) and washed five times in lysis buffer. The fractions eluted with 200 μg ml^-1 3x FLAG peptide (Sigma-Aldrich) were resolved using SDS-PAGE. Specific bands detected by silver staining were excised for in-gel digestion. The digest extracted from the gel was subjected to online HPLC-MS/MS, followed by informatics-based identification of the proteins (Nippon Proteomics).

FlpIn 293 cells (Life Technologies) with low passage number were transfected with empty pcDNA5/FRT vector or pcDNA5/FRT vector containing either wild-type MRP1, wild-type MRP2, or MRP1/MRP2 chimeras and mutants. These vectors were co-transfected with the pOG44 vector according to Life Technologies. This co-transfection resulted in the targeted integration of the expression vector to the same locus in each cell resulting in isogenic cell lines. Transfected cells were grown at 37 °C in 5% CO₂ in DMEM high glucose GlutaMAX (Gibco-Life Technologies) supplemented with 1% penicillin/streptomycin (Sigma Aldrich) and 5% of heat-inactivated fetal bovine serum (PAA, GE Healthcare Life Sciences) and selected with 0.2 mg. mL⁻¹ of Hygromycin B (Life Technologies) for 2 weeks. Phenotyping tests were conducted by observation of characteristic cell morphology, drug responsiveness and expression of MRP1. Passage number of cell lines is between 2-3 times a week for a maximum of 4 weeks. All cell lines were tested for Mycoplasma contamination (Mycoalert Mycoplasma Detection Kit (Lonza).