Figure 3

Analysis of cardiomyocyte polysome RNA. Cardiomyocyte extracts from unstimulated cells (Control) or cells exposed to ET-1 (ET) for 1 h were subjected to sucrose density centrifugation. Fraction 1 is the top and fraction 12 the bottom of the gradient. (a) A₂₅₄ profiles for sucrose density gradients. (b) Agarose gel electrophoresis with ethidium bromide staining of RNA isolated from each fraction highlights 28S, 18S and 5S ribosomal RNA. Fractions 6-11 were pooled for preparation of polysomal RNA for expression profiling. (c) All probesets were used for condition clustering (Pearson complete correlation) of polysome and total RNA prepared from individual sets of samples, and a heatmap of the mean normalized expression for each sample is shown (Log₁₀ scale: cyan = zero, black = 1, red = 6). (d) Principal components analysis identified three components. (e,f) RNAs identified as being significantly changed in the total pool in response to ET-1 at 1 h were clustered according to the profiles in total and polysome RNA giving eight groups with increased expression (e) and four groups with decreased expression (f). Results are means ± SEM for four independent sets of samples. Statistical significance (repeated measures one-way ANOVA with Tukey post-test) p < 0.05 for Control total versus Control polysome (all clusters except PU2, PU5 and PU8), ET-1 total versus ET-1 polysome (all clusters except PU7, PU8, PD3 and PD4).