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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

Fig. 1

ALDH2*2 mutation is associated with increased oxidative stress in patient-derived fibroblasts with familial Alzheimer’s Disease (AD). a) Genotyping of a Japanese AD patient-derived fibroblasts identifies heterozygous ALDH2*2/*1 mutation. ALDH2 protein expression in these cells was measured by immunoblotting three different culture passage numbers. b) The levels of ALDH2 protein were determined in total lysates by immunoblotting of control (healthy subject; H)- and 2*2/*1 AD patient-derived fibroblasts. β-actin was used as loading control. c) ALDH2 protein levels were quantified and represented as fold change of control. d) Enzymatic activity of ALDH2 in lysates from ALDH2*2/*1 AD patient-derived fibroblasts relative to fibroblasts from control was measured over 5 min. e) 4-HNE levels in control and AD patient-derived fibroblasts were measured using 4-HNE assay kit in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. f) Mitochondrial ROS measured by MitoSOX™ in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) cultured in glucose free and galactose supplemented media. g) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. h) Mitochondrial ROS measured using MitoSOX™ in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. i) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. j) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in one control- and one AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. k) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux as in panel j. Data information: Mean, standard deviation, and p-values are shown. Results are presented as fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

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