Figure 4.

Toyocamycin demonstrates CDK9 inhibition in colon cancer cells. YB5 cells were transfected with HA-CDK9 or FLAG-CDK9 vectors. (A) Western blotting shows transfection efficiency. Loading control is shown using β-actin antibody. (B) Percentage of GFP-expressing YB5 cells after DNA hypomethylating drug (DAC, 50 nM, 72 h) or selective CDK9 inhibitor (MC180295, 500 nM, 48 h) in presence or absence of CDK9-expressing vectors (n = 2). (C) Protein expression levels of CDK9 and cell cycle CDK targets. RNA-Pol II (N-term and C-term), phospho-Pol II, Rb, phospho-Rb, and CDK9 levels were measured after 6 h toyocamycin treatment in HCT116 cells (doses are indicated in the graph) or with DMSO. Treatments with CDK9 inhibitor BAY 1251152 at 1–10 µM for 16 h were used as a positive control. β-actin was used as loading control. Molecular weight of each protein is indicated on the graph. Full western blots are available in supplementary. (D) Ratio of Phospho-Pol II Ser 2/Pol II and Phospho-Rb T826/Rb, normalized on β-actin and DMSO are shown (mean  ±  SEM, N ≥ 3 biological replicates, *: p ≤ 0.05 obtained by unpaired Student’s t test). (E) Cell viability analysis by Viacount staining in YB5 and HCT116 cells after 24 and 72 h toyocamycin treatment (0.05, 0.5, 5, and 50 µM, n = 3) relative to untreated cells. (F). Cell cycle analysis by flow cytometry in HCT116 cells after 6 h toyocamycin treatment followed by 18 h without exposure (n = 3, a star indicates statistical difference between treated group and control, unpaired Student’s t-test, p < 0.05).