Figure 3.
NIH3T3 circadian oscillators are functional at significantly reduced RNA polymerase II-dependent transcription rates. (A) NIH3T3 Bmal1-luc cells (grown to confluence) were incubated for 16 h in the presence of 0–5 μg/ml of α-amanitin. At time 0, cells were induced by dexamethasone, α-amanitin was removed and bioluminescence was recorded for 172 consecutive hours (left panel). Increasing doses of α-amanitin resulted in progressively shorter period lengths. The mean period lengths±s.d. determined in ‘n' repetitions in five independent experiments were 25.9±0.9, 25.5±0.3 and 23.6±1.1 h for 0, 1 and 3 μg/ml of α-amanitin, respectively; n=18; see middle panel. The magnitudes (maximal photon counts) decreased with increasing α-amanitin concentrations (right panel). Using bilateral t-test for statistical analysis, the following significance values were obtained for data sets from cells on 3 μg/ml of α-amanitin treatment compared with untreated samples: for period length change P=7 × 10−5; for magnitude P=0.01. (B) NIH3T3 Bmal1-luc cells were incubated for 5 h in the presence of 0–100 nM of actinomycin D. At time 0, cells were treated with dexamethasone, actinomycin D was washed away and bioluminescence was recorded for 186 h (left panel). Increasing doses of actinomycin D resulted in progressively shorter period length: 25.4±0.7, 24.5±0.9, 23.5±0.5 and 23.3±0.7 h for 0, 20, 50 and 100 nM of actinomycin D, respectively; n=5; see middle panel. The magnitudes were reduced by the drug treatment (right panel). The following significance values were obtained for data sets from cells on 50 nM of actinomycin D treatment compared with control: P=0.001; for magnitude P=0.018.