Effects of parental treatment on zebrafish embryo cellular stress response. (A) hsf1, hsp70a, hsp90aa and hsp47 relative gene expression and (B) representative western blot and HSP70, HSP90 and HSP47 relative protein expression in ∼1 h post-fertilization (hpf) embryos derived from adult zebrafish exposed to either control or combined exposure conditions for 14 days. Gene expression data were normalized and expressed as stated in Fig. 2. Statistical differences between gene values were determined by two-tailed t-tests (hsf1: P<0.001, hsp90aa: P<0.001, hsp47: P=0.054) or a Mann–Whitney rank sum test (hsp70a: P=0.004). Representative western blot analysis of HSP70 standard (lane 1), HSP90 standard (lane 2), pool of heat stressed gills (positive control; lane 3), control treatment (lane 4), combined exposure treatment (lane 5) and blank (lane 6). Each western blot run contained multiple samples from each treatment group, but for the sake of presentation clarity, only a single replicate for each treatment is presented, requiring spliced images (represented by dashed lines). Protein expression was normalized to Coomassie stain band intensity and expressed relative to the control treatment for each protein. All statistical differences between protein values were determined by two-tailed t-tests (HSP70: P=0.046; HSP90: P<0.001; HSP47: P=0.374). Differences between treatments for a given parameter are indicated by an asterisk. Data are medians and interquartile ranges in A and are means±s.e.m. in B (hsf1, hsp70a, hsp90aa and hsp47, n=5–7; HSP70, HSP90 and HSP47, n=5–6).
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