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. 2014 Dec 11;9(12):e114899. doi: 10.1371/journal.pone.0114899

Figure 2. Expression profiles of Td'HR3 and Td'E75 mRNA and effects of their dsRNAs on their mRNA levels and the survival rate in firebrats.

Figure 2

Both Td'HR3 (A, C) and Td'E75 (B, D) were rhythmically expressed in LD (A, B) and DD (C, D), peaking late (subjective) day to early (subjective) night (black symbols). DsHR3#1,2 (pink triangles and squares for #1 and #2, respectively) and dsE75#1,2 (green triangles and squares for #1 and #2, respectively) downregulated mRNA levels and disrupted the rhythm of their respective genes (A and B). Treatment with dsDsRed2 as a negative control did not disrupt the rhythmic expression in both genes (A and B, blue symbols), but caused a significant decrease in Td'HR3 mRNA levels at ZT 2 and ZT 18 (+ P<0.05, t test vs intact). White, black, and gray bars above each graph indicate day, night/subjective night, and subjective day, respectively. Total RNA was extracted from firebrats collected at 4-h intervals starting 2 h after lights on or 2 h into subjective day (ZT2 or CT2, respectively). The data collected from 3–4 independent experiments were averaged and plotted as the mean ± SEM values relative to the value of rp49 mRNA used as the reference. Values with different letters significantly differ from each other within the same treatment groups (P<0.05, ANOVA with Tukey test, S1 Table). *P<0.05, **P<0.01, t test vs dsDsRed2 (exact P values are shown in S2 Table). (E) Survival rate after injection of 10.23–13.95 µg/g dsHR3, dsE75, or dsDsRed2 into the abdomen. DsHR3 and dsE75 significantly reduced the survival rate. For further explanation, see text.