Fig. 5

Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)