Isa 2011b

Doxycycline (J60579; Alfa Aesar) was dissolved in diH₂O and SH-SY5Y cells were treated at 3 μg/mL for 72 h. UNC-3230 (5271; Tocris) and ISA-2011B (HY-16937; MedChemExpress) were dissolved in DMSO and cells were treated at 100 nM for 24 h prior to transient transfection or 24 h prior to fixation. Bradykinin acetate salt (B3259; Sigma-Aldrich) was dissolved in diH₂O and perfused at 100 μM in 2 mM Ca²⁺ Ringer’s Solution. Oxotremorine M (O100; Sigma-Aldrich) was dissolved in diH₂O and perfused at 100 μM in 2 mM Ca²⁺ Ringer’s Solution. UTP trisodium salt (U6625; Sigma-Aldrich) was dissolved in diH₂O and perfused at 100 μM in 2 mM Ca²⁺ Ringer’s Solution. Active type 1 recombinant human α-Syn pre-formed fibrils (SPR-322; StressMarq Biosciences) were dissolved in PBS and sonicated for 10 min prior to treatment at 4 μg/mL for 72 h– 14 days. Irrespective of length of α-Syn fibril treatment, all isolated neuronal cultures spent a total of 14 days in culture from initial addition of α-Syn fibrils. α-Syn fibrils were confirmed by immunofluorescence following Triton X- permeabilization and Thioflavin S staining.^{23 (link)}

The full length, truncated and mutated SPNS2 genes, with C-terminal flag tags, were cloned into the pcDNA5/TO plasmid. The plasmid was transfected into HEK 293T cells for 24 h via Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. The SPNS2 overexpressing cells were seeded at density of 1×10⁶ per well in a 6-well plate and were treated with 50 μM ISA-2011B (MedChemExpress) or UNC3230 (Cayman Chemical) for 6 h. After treatment, the cell media were harvested and centrifuged for 20 min at 1,000× g at 4°C and supernatant S1P was measured using a S1P ELISA kit (MyBioSource) and the cell pellets were collected for PI(4,5)P₂ measurement, as described below. For the SPNS2 mutants all constructs were transfected into HEK 293T cells for 24 h via Lipofectamine 2000 (Thermo Fisher Scientific) and seeded at a density of 1×10⁶ per well. After 6 h, the cell media were harvested and used for S1P measurement using S1P ELISA kit (MyBioSource).

Phosphate buffered saline (PBS), medium-199, rat ELISA kits for GH, horse serum, penicillin, and streptomycin were purchased from Life Technologies (Grand Island, NY, USA). Fura 2-AM, the secondary Alexa Fluor 568 donkey anti-rabbit antibody, and 4′,6-diamidino-2-phenylindole (DAPI) were from Invitrogen (Eugene, OR). Rodent LH ELISA Kit was from Endocrine Technologies (Newark, CA). The primary antibody and standard for the PRL radioimmunoassay as well as the rabbit anti-PRL antibody for immunocytochemistry were from the National Pituitary Agency and Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA). ¹²⁵I-Prolactin was from Perkin Elmer Life Sciences (Boston, MA). Bovine serum albumin (BSA) fraction V and saponin were from MP Biomedicals (Solon, OH). The transcriptor first strand cDNA synthesis kit was from Roche Applied Sciences (Indianapolis, IN), Formaldehyde solution was from Thermo Fisher Scientific (Rockford, IL); Bay K8644, PIK93, UNC3230, and Wm were from Tocris (Bristol, UK). ISA2011B was from Med Chem Express (Monmouth Junction, NJ). TRH and dopamine were from Bachem Americas (Vista, CA). Unless stated otherwise, all other chemicals were obtained from Sigma (St. Louis, MO, USA).