Sirt1

Cells were collected and lysed on ice in 6-well plates. BCA protein assay kits were used to measure the concentrations (Thermo Fisher Scientific, Waltham, MA, USA). SDS-PAGE was used to separate the protein samples, which were then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking for 2 h at room temperature with 5% nonfat milk in Tris-buffered solution containing 0.1% Tween 20 (TBST), membranes were incubated overnight at 4°C with primary antibodies (1 : 1000) against Bax (Abcam), Bcl-2(Abcam), Sirt1 (Thermo Fisher Scientific), acetylated FoxO1 (Thermo Fisher Scientific), and β-actin (Abcam). After three washes, the luminescence developed, and the gel imager was exposed for imaging. The strips were analyzed for grayscale using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and processed for semi-quantitative analysis, with three replicates each.

Total RNA was isolated from tissues using TRIzol^® reagent (Thermo Fisher Scientific, Lafayette, CO) or from cells using RNeasy column (Qiagen, Germantown, MD). Reverse transcription was performed using TaqMan reverse transcription reagents (Thermo Fisher Scientific, Lafayette, CO). Specific primers and probes for mouse (P)RR/ATP6ap2, Furin, TFEB, ULK1, LCB3, ATG12, Sirt1, v‐ATPase V1 subunit, β‐actin, and 18s rRNA subunit were purchased from Thermo Fisher Scientific (Lafayette, CO). mRNA expression was quantified by real‐time reverse transcription PCR (qRT‐PCR) using the ABI 7700 Sequence Detection System (Thermo Fisher Scientific, Lafayette, CO). Fold changes in gene expression were determined using the ΔΔC_t method after normalization to β‐actin or 18s rRNA.

CHO-7WD10 and SH-SY-5Y cells were seeded at 1 × 10⁶ cells/well in a six-well plates and stimulated with various concentrations of PGDHC and Pg-LPS as listed for the cytotoxicity assay. After 48 h of stimulation, cells were lysed in the lysis buffer (Thermofisher) and protein concentration was measured using the BCA kit (Pierce). Next, proteins were separated using SDS/PAGE (Bolt 12% gel) electrophoresis, transferred onto a nitrocellulose (NC) membrane and blocked using iBlot2 (Thermofisher). The anti-mouse CT15 polyclonal antibody (1:500) (Calbiochem) was used for detection of full-length APP in CHO-7WD10.
To detect phosphorylated-Tau (p-Tau), sirtuin-1, and cathepsin B in SH-SY5Y cells, rabbit anti-p-Tau (Ser³⁹⁶), and -mouse AT1000 (Thr²¹²/Ser²¹⁴), - Sirt-1 and -cathepsin B polyclonal antibodies (1:1,000; Thermofisher) were used, respectively. The anti-human β-actin antibody (cat # 1:2,000; CST) was used to detect the levels of β-actin as a loading control. Finally, the membranes were washed with tris‐buffered saline (TBS) containing 0.05% Tween 20 and then processed using horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse secondary antibodies (Amersham Pharmacia Biotech) followed by enhanced chemiluminescence detection (ThermoFisher). The signal intensity of Western blots was quantified using Image J.

MicroRNA cDNA preparation kit and primers were purchased from Quanta Biosciences Inc., Gaithersburg, MD, USA. Sirt1 (Thermo Scientific-PA5-23-063, Santa Cruz Biotechnology-sc-15404), Gapdh (Trevigen-2275-PC), eNOS (BD Transduction Laboratories-610297), Stat3 (Cell Signalling Technology-4904), phospho- (Tyr-705)-Stat3 (Cell Signalling Technology-9145), F4/80 (abcam-ab6640) antibodies were used.

All chemicals used in this study were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). GP91, Sirt-1, SOD2, IL-1 beta, NLRP3, Caspase-1 and GAPDH antibodies for performing western blotting analysis were purchased from Thermo Fisher Scientific (Waltham, MA, USA).