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. 2018 Aug 6;7:e36898. doi: 10.7554/eLife.36898

Figure 4. Actinomycin D treatment does not affect the levels of Aurora B or mitotic transcripts.

Figure 4.

(A) Schematic representation of the experimental protocol used to study the effect of ActD in NOC induced mitotic arrested cells by western blot, RNA-seq and qPCR. (B) Western blot analysis of asynchronous (I), G2, and mitotic HeLa cell extracts (Mitosis) with the indicated antibodies. Mitotic extracts were derived from isolated mitotic cells treated cells with DMSO (control), ActD (ActD), cycloheximide (CHX) or Aurora B inhibitor (AurBi). α-tubulin was used as loading control. Approximate molecular weights are shown on the left. (C) Quantification of western blot is depicted in B. The bar represents the mean and the standard deviation from three independent experiments. (D) Normalized expression after RNA-seq of the indicated genes in control or ActD-treated mitotic cells. Error bars represent standard deviations from two independent experiments. (E) Normalized expression after qPCR of human alpha satellite gene in control or ActD-treated mitotic cells (NOC, 1.00 ± 0.01; NOC + ActD, 0.95 ± 0.02, median ±SD from three technical replicates, n.s. p>0.05 relative to control, Mann-Whitney Rank Sum Test). (F) Agarose gel electrophoresis of the qPCR product after amplification of cDNA, obtained from control or ActD-treated mitotic cells, with primers specific for the human alpha-satellite and GAPDH.