Figure 5.

Campothecin and etoposide treatments do not increase global cellular RNA synthesis. Cells were treated for one hour with either vehicle, 2.5 μM of CPT, 50 μM of Eto or 1.25 μM of actinomycin D (ActD), and then further incubated for 30 min with a 0.5 mM ethyl uridine (EU)-containing media. Cells were then fixed and subjected to a click chemistry labeling reaction protocol for analysis. (A) Representative overlay images of EU labeling (green) and nuclei staining (blue) in vehicle-, ActD-, CPT- and Eto-treated cells. (B) Quantitative analysis of the mean fluorescence intensity of individual cells in each condition. Data are displayed as violin plots. Each violin represents the frequency (width) of the mean fluorescence intensity per cell. Data displayed contain values from three independent experiments performed in triplicates and were analyzed using nonparametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test (**** p < 0.0001).