Figure 2

Identification of immediate early and second phase RNAs with increased expression in response to ET-1. Cardiomyocytes were unstimulated (Control), exposed to cycloheximide (CX) alone, or to ET-1 (ET) in the absence or presence of cycloheximide for 1 h (solid bars) or 2 h (open bars). (a) RNAs in CXS1, CXS2 and CXS3 (whose induction by ET-1 was further increased by cycloheximide), and CX1a, CX1b and CX1c (whose induction was not inhibited by cycloheximide) were classified as immediate early gene RNAs. (b) CX2a, CX2b, CX2c and CX2d RNAs showed partial inhibition of the response to ET-1 at 1 h by cycloheximide and are probably second phase RNAs. (c) CX3a and CX3b RNAs were clearly second phase RNAs with >80% inhibition of the response to ET-1 by cycloheximide. The numbers of transcripts in each cluster are shown in parentheses. Statistical significance (repeated measures one-way ANOVA with Tukey post-test) p < 0.05 for Control versus ET-1 (2 h; all clusters), Control versus ET-1 (1 h; all clusters except CXS3, CX1c, CX2d and CX3b), Control versus CX (1 or 2 h; all clusters in (a)), ET-1 (2 h) versus CX+ET-1 (2 h) (all clusters except CX2a/b), ET-1 (1 h) versus CX+ET-1 (1 h) (all clusters except CX1b/c, CX2d and CX3b). (a-c) Results are means ± SEM for three independent sets of samples. (d) Numbers of IEG versus second phase genes upregulated in cardiomyocytes in response to ET-1 over the first 2 h.