Pluronic f 68 non ionic surfactant

The synaptosomal fraction containing both pre- and postsynaptic components was isolated using a well-established method developed in our laboratory [24 (link)–26 (link)]. Briefly, we lysed snap frozen hippocampus (HP) and frontal cortex (FC) tissue from mouse and cognitively intact elderly human brains using SynPER lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) with 1% protease and phosphatase cocktail inhibitors. The brain homogenates were centrifuged at 1200 × g for 10 min at 4 °C. The supernatants (containing the synaptosomes) were collected and centrifuged at 15,000 × g for 20 min at 4 °C. The synaptosomal pellets were resuspended in HEPES-buffered Krebs-like (HBK) buffer (143.3 mM NaCl, 4.75 mM KCl, 1.2 mM MgSO₄·7H₂O, 1.2 mM CaCl₂, 20.1 mM HEPES, 0.1 mM NaH₂PO₄, and 10.3 mM d-glucose, pH 7.4). Finally, 0.5% of Pluronic F-68 non-ionic surfactant (cat# 24040-032, lot# 2275337; Thermo Fisher Scientific) was added to prevent synaptosome aggregation as previously described [27 (link)]. The quality and concentration (synaptosomes/µl) of isolated synaptosomes was routinely verified by flow cytometry and electron microscopy as we previously reported [24 (link)]. Sample processing for the ultrastructure, flow cytometry, and protein analyses are described in detail in the Supplementary Methods (Additional file 1).

NPs were prepared in 20 mg
batches by the nanoprecipitation method using a blend of 15 mg of
PLGA 502H (Sigma) and 5 mg of PEG–PLGA copolymer (5000:10,000
mPEG:PLGA (Akina Inc.)). The polymer was dissolved in 1 mL of acetone
(organic phase) and injected into an aqueous phase containing 0.01%
Pluronic F-68 nonionic surfactant (ThermoFisher Scientific) in a dropwise
manner while stirring. To prepare drug-loaded NPs, 1 mg of RG7388,
Entinostat, or their combination was dissolved in 100 μL of
DMSO prior to addition into the organic phase. The resultant suspension
was stirred overnight to allow acetone evaporation. NPs were then
purified by three wash-spin cycles at 16,000g for
15 min and resuspended in phosphate buffered saline (PBS) for characterization
and cell work. To study nanoparticle uptake, the polymer blend was
modified by adding 1 mg of PLGA-Rhodamine B (lactide:glycolide 50:50)
(Sigma-Aldrich) to 15 mg of PLGA 502H and 5 mg of PEG–PLGA
copolymer, and NPs were prepared using the same method.

STG‐p62 Hap1 cells were grown in suspension at 37°C in a 3 l Wheaton spinner flask for 5 days. The Minimum Essential Medium Eagle (Sigma‐Aldrich) was supplemented with 6 g NaHCO₃, 10% bovine serum (Thermo Fisher Scientific, Waltham), 1% Pluronic™ F‐68 Non‐ionic Surfactant (Thermo Fisher Scientific), 1% MEM Non‐Essential Amino Acids (Thermo Fisher Scientific), 1% GlutaMAX™ Supplement (Thermo Fisher Scientific), Penicillin–Streptomycin (5,000 U/ml; Gibco, Thermo Fisher Scientific). STG‐p62 cells were harvested by centrifugation at 1,300 g for 15 min at 4°C and washed three times with PBS. Pellets were flash‐frozen in liquid nitrogen, resuspended in ice‐cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT supplemented with complete protease inhibitors EDTA‐free cocktail, Roche Diagnostics), and cleared by centrifugation at 13,000 g at 4°C for 15 min. For purification of STG‐p62, 50 μl of StrepTactin Sepharose High performance beads (GE Healthcare) was added to 4 mg total protein in supernatants and incubated for 2 h at 4°C. Beads were washed four times with lysis buffer. Elution was performed in 50 μl of 5 mM biotin in lysis buffer for 60 min at 4°C. The eluted protein was subsequently used undiluted in cluster formation assays.

DHFR sufficient CHO wild type adherent cell line (CHO-WT) was a generous gift from Dr. Gunes Ozhan (Izmir Biomedicine and Genome Center). These cells were grown in DMEM/F-12 medium (11320-033, Gibco) supplemented with 10% FBS (10500064, Gibco) and 1% Penicillin/Streptomycin (15140122, Gibco). DHFR deficient CHO-DG44 adherent cells, obtained as a kind gift from Dr. Lawrence Chasin (Columbia University), were cultured in Alpha MEM Eagle medium (BE12-169F, Lonza) supplemented with 10% FBS and 1% Penicillin/Streptomycin. Both cell lines were maintained at 37 °C and 5% CO₂ cell culture conditions. CHO-DG44 suspension cells, adapted in-house from CHO-DG44 adherent cells to grow in serum-free suspension culture, were cultivated in CD DG44 medium (12610-010, Gibco) supplemented with L-glutamine at 8 mM final concentration and 18 ml/L (1.8%) Pluronic F-68 Non-ionic Surfactant (24040032, Gibco), and maintained at 37 °C and 8% CO₂ cell culture conditions and passaged every 3–4 days by dilution. Cells were routinely tested negative for mycoplasma contamination using a PCR-based mycoplasma detection kit (G238, ABM).

All constructs were N-terminally tagged with 10xHis followed by EGFP (Ca²⁺ imaging) or mVenus (cryo-EM)^{83 (link)} followed by a human rhinovirus 3 C protease^{84 (link)} cut-site and then human type 3 IP₃R. Plasmids were transformed into DH10Bac cells to generate bacmids^{29 (link)}. 100–200 µg of purified bacmid were incubated with 400 µg of 25,000 MW polyethyleneimine (PEI; Polysciences Cat# 23966) in 1.4 mL water at 55 °C for 30 minutes to sterilize, then added to 50 mL of Sf9 cells at 1×10⁶ cells/mL grown in suspension at 27–30 °C. The Sf9 TNM-FH (Grace’s modified) media was supplemented with 1% penicillin/streptomycin, 0.1% Pluronic F-68 nonionic surfactant (Gibco Cat# 24040), and 4-8% fetal bovine serum to stabilize the virus. Virus titer was amplified to P3 and separated from cell debris by centrifugation. P3 virus was used to infect mammalian HEK293S GnTI^- (ATCC CRL-3022) cells at a density of 3×10⁶ cells/mL at a ratio of 50 mL virus for 800 mL cells and simultaneously stimulated with 4.5 mM valproic acid (VPA; Sigma Cat# P4543). Pellets were harvested from cells by centrifugation at 48–72 hours after infection and snap frozen.