Pre-treatment with ActD protects zebrafish embryos from MLN4924-induced toxicity.

(a) Zebrafish embryos were treated with the indicated doses of MLN4924 for 24 hrs before cell extracts were used for western blot analysis (cropped blot for GAPDH). (b) Similar experiment as in (a) except embryos were pre-treated with the indicated doses of ActD 4 hrs before MLN4924 addition. Extracts were analysed by western blotting for the indicated proteins (cropped blots for caspase-3, GAPDH). (c) Similar experiment as in (b) and extracts were used to measure cell viability and caspase-3/7 activity. Values represent the average of 3 independent experiments as fold change in caspase-3/7 activity normalised to cell viability, +/−S.D. as error bars. (d) Zebrafish embryos were treated as in (b) and used for immunofluorescence analysis with active caspase-3 antibody. Selected areas (white boxes-enlarged insets) were used to quantify the active caspase-3 signal, represented in (e) as arbitrary units of intensity, n = 25, error bars +/−S.D.