Fig. 7

PolyQ expansion affects p53-dependent apoptotic function in cells and in zebrafish.

(A) HCT116 p53^+/+ and HCT116 p53^-/- cells transiently transfected with Flag-vector or normal Flag-ATX-3 or Flag-ATX-3-80Q were treated with CPT (1 μM) for 24 h and then analyzed by flow cytometry for apoptosis using Annexin-V/PI. Results are the mean ± SEM of three independent experiments. * denotes p < 0.05. Cell lysates were immunoblotted with indicated antibodies. Underlying data are shown in S1 Data. (B) HCT116 p53^+/+ and HCT116 p53^-/- cells transiently transfected with Flag-vector or the normal Flag-ATX-3 or Flag-ATX-3-80Q were treated with CPT (1 μM) for 24 h. Lysates and proteins in the medium were analyzed by immunoblotting with indicated antibodies. M, released into medium; P, pellet. (C and D) Flag-ATX-3-80Q induced p53-dependent apoptosis in zebrafish. Zebrafish embryos were injected with 100 pg of Flag-ATX-3 (ATX-3) or Flag-ATX-3-80Q (ATX-3-80Q) mRNA at one-cell stage. UIC, uninjected control. Twenty-four hpf, embryos were subjected to TUNEL assay and observed by confocal microscopy. Endogenous p53 levels in the p53 WT and p53 mutant zebrafish were examined by western blot. (C) TUNEL-positive cells were quantified from 5~7 embryos each. Results are the mean ± SEM. ** denotes p < 0.01, compared with the control. (D). Underlying data are shown in S1 Data. (E) Whole-mount in situ hybridization analyses of the midbrain neural marker otx2 in uninjected control (UIC) or normal ATX-3 (WT) or ATX-3^exp (80Q) mRNA-injected p53 WT (left) and p53 mutant (right) zebrafish embryos at 24, 48, and 72 hpf. Embryos were shown in lateral views with anterior to the left. The ratio of embryos with the representative phenotypes was indicated. Both WT and 80Q mRNA injections resulted in obvious otx2 signal decreases in p53 WT but not p53 mutant zebrafishes, with more profound otx2 signal reduction in 80Q mRNA injections. MB denotes midbrain.