Fig. 5: AJ-5 triggers the intrinsic and extrinsic apoptosis pathways in RMS cells.
a Representative light microcopy images (×200; EVOS XL AMEX1000 Core Imaging System) showing the morphology of RH30 and RD cells treated with vehicle, 0.1 µM or IC50 AJ-5 for 24 and 48 h. Numbered circles correspond to magnified images on the right, which highlight characteristic apoptotic morphology including membrane blebbing and cell shrinkage. b Western blot analyses of protein harvested from cells treated as in a and incubated with antibodies as indicated. p38 was used as a loading control and densitometry readings were obtained using ImageJ. Protein expression levels are represented as a ratio of protein of interest/p38 normalized to vehicle control samples (where possible). Blots are representative of at least two independent repeats. Broken lines indicate where lanes not relevant were removed. c Caspase-Glo assays showing the enzymatic activity of caspase-3/7, caspase-8 and caspase-9 for cells treated with vehicle or IC50 AJ-5. Doxorubicin was included as a positive control for caspase-8 and -9 assays at the IC50 concentration (0.5 µM) established in the cell lines tested. Graphs represent the mean fold change of caspase activity ± SEM pooled from three independent repeats. Data were analysed using GraphPad Prism 6.0 and a parametric unpaired t-test was performed where *p < 0.05, **p < 0.01, ***p < 0.001
