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. 2019 Jul 25;20(9):e47592. doi: 10.15252/embr.201847592

Figure 4. A tight interplay between CDK12 kinase activity, expression of DNA replication genes, cell cycle progression, and genome stability.

Figure 4

  • A
    Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum‐containing medium with (+) or without (−) 3‐MB‐PP1. 3‐MB‐PP1 was washed away and replaced with fresh medium at indicated times after the release and samples were subject to RT–qPCR, Western blotting, and flow cytometry analyses at 7, 12, and 15 h after the release, respectively. Note that shown wash away time points (2, 3, 4, 5 h) are valid for RT–qPCR only, for Western blotting and flow cytometry 1, 2, 3, 5 h and 1, 3, 5, 7 h wash away time points were applied, respectively. All experiments were performed in at least three replicates.
  • B–D
    Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT–qPCR (B), Western blotting (C), and flow cytometry analyses (D) of replication gene mRNA, protein levels, and cell cycle progression, respectively. RT–qPCR, Western blotting, and flow cytometry analyses were performed 7, 12, and 15 h post‐release, respectively. CTRL = control samples without the 3‐MB‐PP1. In B, n = 3 and error bars indicate SEM. In (C, D) representative images from three biological replicates are shown.
  • E
    Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum‐starved AS CDK12 HCT116 cells treated with 3‐MB‐PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions, and studied DNA replication factors are indicated. A representative image of three replicates is shown.
  • F
    Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3‐MB‐PP1, and replicating BrdU‐stained cells were quantified by FACS analyses. CTRL = control samples without the 3‐MB‐PP1. A representative image of three replicates is shown.
  • G, H
    Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3‐MB‐PP1 (24 or 48 h), 4 mM hydroxyurea (5 h), or control solvent (CTRL) were identified by microscopy. A representative image from three biological replicates is shown (G). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in (G) are quantified (H).

Source data are available online for this figure.