Poly d lysine coated

For single-molecule and FRAP microscopy experiments, 8-chamber Nunc Lab-Tek #1.0 borosilicate coverglass systems were used (cat#: 155411, Thermo Fisher Scientific). The chambers were sterilized with 70% isopropanol and coated with fibronectin bovine plasma (cat#: F1141, Sigma-Aldrich) for 20 min at 37 °C. Thereafter, the chambers were washed twice with phosphate-buffered saline (cat#: D8537; Sigma-Aldrich). The cells were detached with accutase solution (cat#: A6964, Sigma-Aldrich) for 4 min at 37 °C; 50,000 cells resuspended in 500 µL cell culture medium were then seeded into each well. After 12–16 h, the cell culture medium was replaced with imaging buffer (2% FBS in Hanks’ Balanced Salt Solution [cat#: H8264, Sigma-Aldrich]) and the samples were measured.
For confocal microscopy, transfected CHO cells were seeded into a poly-D-lysine-coated (0.05 mg/mL; cat#: P1149, Sigma-Aldrich) 29-mm glass-bottom dish with 20-mm micro-well #1.5 cover glass (cat#: D29-20-1.5-N, Cellvis, Mountain View, CA, USA) at a density of 100,000 cells per dish. The following day, the culture medium was removed and the cells were incubated for 10 min with 10 nM of the nisoxetine-based fluorescent probe AC1-146^{56 (link)} diluted in Krebs-HEPES buffer (KHB: 25 mM HEPES, 120 mM NaCl, 5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, and 5 mM D-glucose, pH 7.3) at room temperature.

Cortical progenitor cells were isolated from E12.5 mice. Isolated cortices were transferred to Neurobasal medium (Thermo Fisher Scientific) containing 40 ng/ml fibroblast growth factor 2 (Promega), 2% B27 (Thermo Fisher Scientific), 120 mg/ml penicillin, 200 mg/ml streptomycin sulfate, and 600 mg/ml glutamine. The cells were plated on poly-D-lysine-coated (Sigma) and laminin-coated (BD) 4-well chamber slides at a density of 4.0 × 10⁵ cells/well and were maintained at 37 °C in the presence of 5% CO₂. For transfections, medium was mixed with 2 μg of plasmid DNA or shRNA vector and 1.5 μl of FuGENE HD (Promega) in 100 μl per well for 15 min at room temperature, after which the transfection mixture was applied to the chamber slides.

Mouse cerebral cortices were extracted from neonatal P0-1 pups and placed into an ice-cold HBSS/FBS solution: Hank’s Balanced Salt Solution (Sigma, St. Louis, MO), 4.2 mM NaHCO₃, 1 mM HEPES, and 20% FBS. The tissue was washed three times with HBSS and then digested at 37°C/15 min with Papain (Worthington Biochemical, Lakewood) in a solution that contained 137 mM NaCl, 5 mM KCl, 7 mM Na₂HPO₄, and 25 mM HEPES (pH 7.2). The tissue was washed 3 times with 20% FBS, three times with HBSS, and three times with growth medium containing Neurobasal A, B27, Glutamax and Penicillin/Streptomycin (Invitrogen, Carlsbad, CA). Tissue was then triturated in growth medium containing 50 units of DNAse I (Invitrogen, Carlsbad, CA). The neurons were pelleted by centrifugation (600 × g for 10 min) and plated at a density of 40,000 cells/cm² on poly-D-lysine-coated (Sigma-Aldrich, St. Louis, MO) 60 mm dishes. Neurons were incubated at 37°C/5% CO₂, and half of the medium was replaced with fresh medium once a week. 0.5 µM dexamethasone (Sigma-Aldrich, St. Louis, MO) or vehicle (EtOH) was applied after 14 days in culture for 7 days.

The protocol for generating amygdala neuronal cultures was adapted from protocols for hippocampal neuron cultures.^{24 (link), 25 (link)} Briefly, rat amygdala was dissected from E19 embryos and placed in Calcium-free Hank's Balanced Salt Solution (HBSS; Invitrogen) containing 1% papain for 20 min, triturated in Basal Media Eagle (Invitrogen) supplemented with B-27 (Invitrogen), and plated at 100,000 cells per milliliter in Neural Basal (NB) (Invitrogen) supplemented B-27, 1% penicillin and streptomycin (Invitrogen), and 1% glutaMAX (Invitrogen) on poly-D-lysine-coated (Sigma-Aldrich) coverslips in 24-well plates. Cells were maintained at 37°C, 5% CO₂, 95% humidity. The medium was partially changed weekly.

These procedures were performed as previously described [14 (link), 28 (link)]. Cells were plated on poly-D-lysine-coated (0.05% w/v; Sigma) 8-well glass culture slides (BD Biosciences) and cultured (48 h) in RPMI 1640 (growth medium) prior to CDDP treatment. For immunostaining, cells were fixed in paraformaldehyde (4%, 1 h, RT), washed in PBS, and blocked with 1% BSA. Mitochondria were visualized by immunofluorescence microscopy, using a mouse monoclonal antibody anti-human Tom 20 (1:100; Santa Cruz Biotechnology) and Alexa Fluor 488 goat anti-mouse secondary antibody (1:500; Invitrogen). Confocal images were obtained (× 100 objective) on an Olympus IX81 inverted microscope with appropriate argon lasers (488 nm). Mitochondrial phenotype of each cell was categorized as being tubular, intermediate or fragmented, as previously described [14 (link)]. At least 100 cells were analyzed per treatment group.

C-kit-positive murine CSCs were isolated using our previously described methods [15 (link)]. Briefly, CSCs were obtained from the hearts of 2-month-old wild-type male C57BL/6 mice (Yangzhou Laboratory Animal Center). After the CSCs were cultured for 1 to 2 weeks, a layer of fibroblast-like cells migrated from the adherent myocardial tissue. Some small, round, phase-bright cells emerged from these fibroblast-like cells and were collected using the digestion enzyme Accutase (Millipore, USA). Enriched c-kit-positive cells were further isolated using c-kit magnetic-activated cell sorting (MACS) magnetic beads (Miltenyi Biotec Inc., Germany) according to the manufacturer’s instructions. These separated cells were seeded on poly-d-lysine-coated (Sigma, USA) dishes at a density of 2 × 10⁴ cells/ml in cardiosphere growth medium (CGM: 65% Dulbecco’s modified Eagle’s medium (DMEM)-F12 (HyClone, USA) containing 10% fetal calf serum (Gibco, USA), 2 mmol/l l-glutamine (Gibco, USA), 0.1 mmol/l 2-mercaptoethanol (Sigma, USA), 2% B27 (Gibco, USA), 5 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, USA), 10 ng/ml epidermal growth factor (EGF; Peprotech, USA), 40 nmol/l cardiotrophin-1 (Peprotech, USA), 1 unit/ml thrombin (Sigma, USA), 100 U/ml penicillin G (Gibco, USA), and 100 mg/ml streptomycin (Gibco, USA)). The culture medium was changed every 3 days.

HEK293 cells were transfected using a linear polyethylenimine (PEI) derivative, the polycation ExGen500 (Fermentas Inc., Hanover, MD, USA) as previously reported (eight equivalents PEI/3.3 μg DNA/dish)⁶¹ . Co-transfection was performed using the ratio for GFP-fused TRPP3 (WT or either mutant R278Q, R378W, or R278Q/R378W), and PKD1L3 cDNA constructs of 1:1. For testing possible dominant negative action of the double mutant, cells were co-transfected with WT, R278Q/R378W TRPP3-GFP and PKD1L3 cDNA constructs with a ratio of 1:1:2. Transfected cells were incubated for 24 h and seeded on 35 mm diameter dishes Poly-d-Lysine-coated (Sigma). Experiments were performed 48 h post-transfection at room temperature (22–24 °C).