Cell lysis buffer

Cells were lysed in Cell Lysis Buffer (New England Biolabs) supplemented with Complete Protease Inhibitors (Roche) and subjected to SDS-PAGE, and transfer to Immobilon-P membrane (Merck-Millipore). After blocking in the blocking buffer (TBST + 5% non-fat milk), primary and HRP-conjugated secondary antibodies (Cell Signaling catalogues 7074S and 7076S) were applied in the blocking buffer and antigens were revealed with SuperSignal West Pico chemoluminescent reagent (ThermoFisher). Antibody to AK2 (HPA018479) was from Cambridge Biosciences. Antibody to c-MYC (clone Y69—ab32072) was from Abcam. Anti-phospho-AMPK^Thr172 (Cell Signaling catalogue #2535), anti-AMPK (Cell Signaling catalogue #5831), anti-β-actin (13E5—Cell Signaling catalogue #4970S), and for Fig. 3E anti-c-MYC (clone 9E10—Novus Biologicals #NB600-302).

The fragments of lncR-125b and IGF2 containing an miR-125b binding site were amplified and subcloned into the XhoI and NotI sites of the psiCHECK-2 vector (Promega), named lncR-125b-wild and IGF2-wild. The mutant derivatives (lncR-125b-mut, IGF2-mut), accessed by altering the miR-125b binding sites and generated using MutanBEST Kit (Takara), were also cloned into the psiCHECK-2 vector. Mutant plasmids were generated by changing the binding site of miR-125b from CTCAGGG to GTCCATA (lncR-125b-mut), or from TCAGGGA to CACATAA (IGF2-mut). The sequences of wild-type and mutated sequences are presented in Figure S1. Primers used for plasmid construction are listed in Table S2. For the luciferase reporter assays, SMSCs (∼1 × 10⁴ cells per well) were cultured in GM in 24-well plates, with conditions similar to those for SMSCs, and transfected when cells reached 80%–90% confluence. GM was completely replaced by DM at 6 h post-transfection. The transfected cells were harvested and lysed with Cell Lysis Buffer (NEB, Ipswich, MA, USA) and luciferase activities were measured 48 h after differentiation using a Dual-Luciferase^® Reporter Assay System (Promega).

The mitochondrial fraction was isolated from the LV tissue of two mice per group using the Mitochondrial Isolation Kit for Tissue (Fisher Scientific) according to the manufacturer's protocol. The mitochondrial pellet was then lysed with ice‐cold cell lysis buffer (New England Biolabs) and western blotting was used to measure mitochondrial lysine acetylation.

NCM460 cells were seeded at 20,000 cells/cm² in a 6-well plate and grown for 3 days in RPMI media supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a 95% humidified atmosphere with 5% CO₂. Medium was removed and replaced with DMEM supplemented with 1% NEAA containing or not containing the STW extracts. After 4 h incubation, the medium was removed and cells were induced for various times with human CM consisting of 10 ng/ml TNF-α, 5 ng/ml IL-1β, and 10 ng/ml IFN-γ. Cells were washed with PBS, incubated with cell lysis buffer (New England Biolabs) for 5 min and collected. Lysated cells were sonicated four times for 5 min and centrifuged at 14,000 × g for 10 min. The supernatants were used for further analysis.

Frozen substantia nigra tissue was selected from the same Control (N = 3), PSP (N = 3) and PD (N = 3) cases used for OPAL multiplex immunofluorescence. Protein lysates were generated using cell lysis buffer (NEB) and brief sonication on ice, followed by centrifugation to pellet insoluble material. Co-immunoprecipitation was carried out using the Dynabeads Protein G immunoprecipitation kit (ThermoFisher Scientific), with an anti-α-synuclein antibody (Abcam) as bait. Proteins bound to beads were eluted and assayed by western blot (as described above) and probed with an anti-LRRC37A antibody (ThermoFisher Scientific). Whole protein lysate and IgG only controls were run on the same blots.

Monolayers of human myometrial cells were lysed in cell lysis buffer obtained from New England BioLabs (UK) Ltd., scraped and collected in Eppendorf tubes. Samples were centrifuged at 13,000g for 15 min. at 4°C. Supernatant were then transferred and stored at -80°C.