Fig 1: ADS024 inhibited caspase three cleavage in toxin-treated human colonic epithelial cells. (A,B) Apoptosis array. Serum-starved primary human colonic epithelial cells were pretreated with ADS024 ethyl acetate extract at 1X or sterile filtrate at 0.0001X dilution for 30 min, followed by toxin B (0.1 μg/ml). A total of 24 h later, the cells were collected for Proteome Profiler Human Apoptosis Array (ARY009, R&D Systems). (A) Bio-Rad ChemiDoc Imaging system captured the images. The rectangles highlighted the inactive procaspase three and active cleaved caspase three. The images are representative of three independent experiments. (B) Bio-Rad Image Lab Software performed quantitation of cleaved caspase 3/procaspase 3 signal. Toxin B increased cleaved caspase 3/pro-caspase 3 ratio, which was reduced by ADS024 EA extract 1X and sterile filtrate 0.0001X dilution. (C) Cleaved caspase 3 ELISA. Serum-starved primary human colonic epithelial cells were pretreated with ethyl acetate extracts of ADS024 and DSM7 at 1X or sterile filtrates of ADS024 and DSM7 at 0.0001X dilution for 30 min, followed by the addition of toxin B (0.1 μg/ml). The cells were lysed by RIPA buffer with 1X PIC and 1X EDTA. The cleaved caspase three levels in cell lysates were measured by ELISA. (D) Apoptosis assays. Serum-starved NCM460 cells were pretreated with ethyl acetate extracts of ADS024 and DSM7 at 1X and sterile filtrates of ADS024 and DSM7 at 0.0001X dilution with or without adding 10 μM procaspase activating compound 1 or PAC1 (#10009317, Cayman Chemical). PAC1 was used to activate caspase three cleavage. Reagents of Promega RealTime-Glo Annexin V apoptosis assay in a 1:1,000 ratio were added simultaneously. Thirty minutes later, toxin A or toxin B (0.1 μg/ml) was added to start the apoptosis process. After 24 h, the luminescent (apoptosis) signals were read by a 96-plate reader. The Annexin V luminescent signal above 100% indicated the occurrence of apoptosis. The ADS024 EA extract-mediated inhibition of toxin A- and B-dependent apoptosis was reversed by PAC1 pretreatment. The ADS024 sterile filtrate-mediated inhibition of apoptosis in toxin B-treated cells was reversed by PAC1 pretreatment. Results were pooled from 3–4 independent experiments. One-way ANOVA tests were used.