human taurd p301s fret biosensor  (ATCC)

Journal: bioRxiv

Article Title: Ketone body β-hydroxybutyrate restores neuronal Tau proteostasis via ketolysis-independent mechanism

doi: 10.64898/2026.01.30.702936

Figure Lengend Snippet: (A) Schematic depicting enantiomer strategy for dissecting ketolysis-dependent and -independent features of βHB. AcAc, acetoacetate. (B) Climbing score for diet-fed WT and hTau+ flies at 7, 14, and 21 days old. Data represents the average scores of 4 vials per group from three trials. n = 15 flies/vial. AUC, area under the curve. (C) Survival curve for diet-fed WT and hTau+ flies. n = 20 flies/vial, 5 vials per group. (D) Schematic of organotypic brain slice culture (BSC) paradigm used in E-G. PFF, pre-formed fibrils. (E-G) Representative immunofluorescent images (E) and quantification (F, G) of BSC treated with AAV-Tau P301S , K18-Tau P301L PFF, and βHB or NaCl for 10 days, stained for MC1 (magenta) and DAPI (white). Each point represents an individual brain slice, with slices in the same well stacked into one column. Thick, color-coded bars represent the well mean (n = 4-6 slices/well), and black bars represent the overall group mean ± SD (n = 3 wells, from separate batches). Each batch was normalized to the average of its respective control (NaCl) well. Scale bars: 500 μm. (H) Schematic of primary neuron Tau secretion paradigm for 4I. (I) Quantification of percent of Tau secreted into the conditioned media (CM) in primary neurons treated with various metabolites, with or without glucose, for 1 hr. Value calculated by the amount of Tau in the CM divided by the sum of the CM Tau and intracellular (lysate) Tau, and each point represents one independent well, normalized to the control (NaCl + glucose) well/s from its respective plate. Tau levels were measured using ELISA. aCSF, artificial cerebrospinal fluid. (J) Quantification of relative intracellular Tau levels in primary neurons, corresponding to 4I. Each point represents one independent well, normalized to the control (NaCl + glucose) well/s from its respective plate. (K) Lactate dehydrogenase (LDH) assay as a proxy for cytotoxicity. 100% represents the amount of LDH within the media upon complete cell lysis/death. (L) Schematic of HaloTag-Tau P 301 L turnover assay in induced pluripotent stem cell-derived neurons (i 3 Ns). i 3 Ns do not express 4R-Tau normally, so an anti-4R-Tau antibody identifies total HaloTag-Tau. TMR, tetramethylrhodamine-conjugated Halo ligand. (M-N) Representative Western blot (M) and quantification (N) of Halo-Tau in-gel fluorescence. Each point represents one independent well, normalized to the average of the NaCl-treated values for its respective plate. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns not significant by AUC analysis (B, C), linear mixed-effects model with Tukey post-hoc test (F, G), and Dunnett’s multiple comparison test (J-K, N). For I, **** p < 0.0001, ns not significant compared to NaCl + glucose, #### p < 0.0001 compared to NaCl - glucose group by Šídák’s multiple comparisons test.

Article Snippet: Human TauRD P301S FRET biosensor-expressing HEK293T cells (ATCC CRL3275) were maintained and passed in complete DMEM media (DMEM with 10% FBS, 100 μg P/S).

Techniques: Slice Preparation, Staining, Control, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Lysis, Turnover Assay, Derivative Assay, Western Blot, Fluorescence, Comparison