Prl tk dna

Cell culture medium and all the other materials required for cell culture were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). LPS (Escherichia coli O55:B5), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), and the specific protein kinase inhibitors (PD98059 and SP600125) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CellTiter⁹⁶ AQ_ueous One Solution Cell Proliferation assay kit, dual-luciferase assay system, murine NF-κB promoter/luciferase DNA, pRL-TK DNA, and moloney murine leukemia virus (M-MLV) reverse transcriptase were obtained from Promega (Madison, WI, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β, and IL-6 were obtained from eBioscience (San Diego, CA, USA) and PGE₂ ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). Primary and secondary antibodies were purchased from Cell Signaling Biotechnology (Danvers, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. 4’,6-Diamidino-2-phenylindole (DAPI), Lipofectamine Plus Reagent, TRIzol, and Alexa Fluor® 488-conjugated secondary antibody were purchased from Invitrogen (Carlsbad, CA, USA). The enhanced chemiluminescence (ECL) detection kit was purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA).

The activity of the FOXO transcription factor was assessed using the pGL-3 × DBE plasmid (Promega)^{43 (link)}, which incorporates three iterations of FOXO-responsive promoters and encodes firefly luciferase. Concurrently, the pRL-TK plasmid (Promega), expressing renilla luciferase constitutively, was transfected as an internal control. Astrocytes were seeded in 60 mm diameter plates (1 × 10⁶ cells/plate) and transfected after 48 h (approximately 60% confluence) using Fugene 6 (Promega) with 1 µg DBE DNA, 0.2 µg pRL-TK DNA, and a DNA: Fugene ratio of 1:6. Following 48 h of transfection, the cells were treated by washing twice with phosphate buffered saline (PBS) and incubating for 24 h in DMEM containing either 4.5 g/L or 1 g/L glucose concentration, with or without 1 nM Klotho. Post-treatment, the cells were lysed as per the Dual-Luciferase^® Reporter Assay system (Promega) guidelines. Equal sample volumes were used, and the luminescence was developed and read according to the manufacturer's instructions (Promega) on opaque white plates using a Synergy H1 Hybrid Multi-Mode plate reader (BioTek). The results are presented as the ratio of firefly luciferase fluorescence to renilla luciferase, normalized to the mean of the control group.