60 mm dish

ARPE-19 cell apoptosis was evaluated by propidium iodide (PI) staining with the flow cytometry analysis. Briefly, 2 × 10⁵ cells were seeded on a 60-mm dish (Corning Life Sciences) and treated with nicotine and/or cotinine for 7 days. Cells in the supernatant were collected every day when the medium was changed, and they were immediately fixed with 70% ethanol at 4 °C for at least 24 hours. Together with the trypsinized cells at Day 7, the fixed ARPE-19 cells were treated with 50 μl of 100 μg/ml RNase A (Pure link^TM, Invitrogen) and 950 μl of 50 μg/ml PI (Sigma-Aldrich) in PBS for 20 mins at room temperature in dark. The cells were then passed through a 35 μm cell strainer (Falcon, Corning, NY). 1 × 10⁵ cells for each sample were analyzed by the flow cytometry machine (Cytomics FC500; Beckman Coulter, Indianapolis, IN). Single cells were identified using forward scatter (FS) and side scatter (SS). Cell clumps or doublets were excluded using the pulse area versus the peak height. The distributions of different cell cycle phases were identified using the cell counts (excitation: 488 nm, emission: 620 nm) versus the intensity of PI.

Cells were seeded in a 60-mm dish (Corning Inc., Corning, NY, USA) at a density of 6 × 10⁵ cells for cystine starvation. The cells were lysed with 5% 5-sulfosalicylic acid solution. The cellular level of glutathione was determined using a Glutathione Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol.

A total of 2.5 × 10⁶ ARPE-19 cells (ATCC, Manassas, VA) were seeded in a 60-mm dish (Corning, Tweksbury, MA) and cultured with Dulbecco’s Modified Engle’s Medium/F12 (Thermo Fisher, Waltham, MA) plus 10% of bovine serum albumin (Thermo Fisher) as well as 1% penicillin/streptomycin (Thermo Fisher) in a 37 °C humidified and 5% CO₂ culture incubator. The cells were maintained in the 6 wells or harvested with trypsin (Thermo Fisher) and then seeded in 96 wells (Corning) before specific experiments.

Mouse embryonic neural stem cells were isolated from E14 cortices using NeuroCult Proliferation Kit Mouse (STEMCELL Technologies) and maintained as recommended. Primary neurons were prepared from E15.5 mouse cortices and cultured essentially as in ref. 58 (link), except no astroglial feeders were used for cultures maintained ≤7 days in vitro (DIV). Neurons were transfected using NeuroMag reagent (Oz Biosciences) as recommended. Briefly, 2.4 × 10⁶ cortical neurons were plated per 60-mm dish (Corning) pretreated with poly-D-lysine (Sigma). At DIV2–DIV5, 0.1 μg of TTP expression plasmid (pBS-CMV-TTP) or corresponding vector control or 4 μg of a dTomato reporter plasmid (dTom-3′HuR-pA2mut or dTom-3′HuR-pA2mut/ΔARE) was incubated with 6 μl NeuroMag beads in 150 μl Opti-MEM I for 20 min and the mixture was added dropwise to the dish containing neurons. The cultures were then incubated on top of Super Magnetic Plate (Oz Biosciences) for 40 min and incubated for another 40–48 h at 37 °C and 5% CO₂ before subsequent analyses.

We selected two squamous cell carcinoma cell lines (SAS and Ca9-22) that were highly sensitive to NTP and one non-tumorous fibroblast line (IMR-90-SV). In the NTP group and the Ar alone group, a total of 1.5 × 10⁶ cells/well were seeded in a 60-mm dish (Corning, Corning, NY) and cultured for 24 h. NTP or Ar alone was applied, and the cells were incubated for an additional 24 h. After incubation, we used the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s protocol (Nacalai Tesque) and evaluated cell death using the Gallios flow cytometer (Beckman Coulter, Brea, CA). Furthermore, we performed a TUNEL assay. In the NTP group and the Ar alone group, a total of 5,000 cells/well were seeded in a 96-well plate (Thermo Fisher Scientific) and cultured for 24 h. NTP or Ar alone was applied, and the cells were incubated for another 24 h. After incubation, we used the In Situ Cell Death Detection Kit, Fluorescein to observe apoptosis microscopically according to the manufacturer’s protocol (Roche Applied Science, Mannheim, Germany) and observed the staining results, using the BZ9000 microscope (Keyence, Osaka, Japan).