Figure 3.

The effect of TPH104c on the levels of apoptotic and anti-apoptotic proteins in BT-20 cells. (a) Representative images featuring morphological changes in BT-20 cells (under 20× magnification) after incubation with vehicle (0 μM, media without drug), 0.1, 0.3, or 1 μM of TPH104c for 0, 24, 48 or 72 h. (b) Representative images of BT-20 cells with vehicle (0 μM), 2, or 5 μM of TPH104c for 24 h or paclitaxel (PTX, 1 μM, a positive control) and stained with Hoechst 33342 dye. TPH104c did not produce condensed or fragmented nuclei compared to cells incubated with paclitaxel (PTX). Scale bar = 25 μM. (c) Western blot images representing the levels of the apoptotic molecules, cleaved caspase-3, caspase-3, cleaved caspase-7, caspase-7, cleaved caspase-9, caspase-9, cleaved caspase-8, caspase-8, BAX, BAK, BCL-2, cleaved PARP and PARP, following incubation with vehicle (0 μM), 0.5, 1, 2 or 5 μM of TPH104c. The proteins are expressed as a ratio to β-actin, followed by normalization to the vehicle control. (d) The level of each protein is shown by histograms. Clvd = cleaved; Csp = caspase. The data represent the average ± SEM of four separate studies. (e) Caspase-Glo 3/7 assay results are represented as a bar graph and curve, showing a decrease in the levels of caspase-3 and caspase-7 by TPH104c, in a concentration-dependent manner in BT-20 cells, after 24 h of incubation. In contrast, 1 µMof PTX induced caspase- 3 and 7 activity (n = 2). (f) The IC₅₀ values, using the MTT assay, for TPH104c in BT-20 cells that were preincubated with zVAD-FMK (a pan-caspase inhibitor) and then incubated with varying concentrations of TPH104c for 72 h. The data were obtained from three independent experiments conducted in triplicate and represent the average ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns means non-significant. Original Western Blot images can be found in Supplementary Materials.