Fig. 5: Evaluation of the anti-apoptotic effects of AC102 in ethanol-challenged HT22 cells in vitro.

a Schematic representation of cell damage and apoptosis assays. HT22 cells were treated for 5 h in cell culture medium only (Control), medium containing 4.5% ethanol (EtOH) or co-treated with varying concentrations of AC102 from 1 µM to 100 µM. Cell damage was quantified by changes in cell morphology (round cells), and caspase-dependent apoptosis was assessed by quantifying caspase-3/7 activity using a commercial kit as well as expression of cleaved caspase-3 by immunohistochemistry. b Percentage (%) of round cells following EtOH challenging with co-treatment of AC102 at 1, 10, 30 and 100 μM (n = 3). c Confirmation of AC102’s anti-apoptotic effects by quantification of caspase-3/7 activity using the Caspase-Glo® 3/7 assay. Arbitrary relative luminescence units (a.u.) were measured after a 5 h incubation in culture medium, 4.5% EtOH and added 30 or 100 μM of AC102 (n = 7). d Exemplary images of HT22 cells following the incubation with culture medium, added 4.5% EtOH and the additional 30 and 100 μM of AC102 (red asterisks indicate round damaged cells, scale bar = 20 µm). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.