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

Figure 2. CDK12 kinase activity is essential for optimal G1/S progression independently of DNA damage cell cycle checkpoint.

Figure 2

  • 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. DNA content was analyzed by flow cytometry at indicated time points after the release.
  • B
    CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation. Flow cytometry profiles of control (−3‐MB‐PP1) or inhibitor (+3‐MB‐PP1) treated cells from the experiment depicted in Fig 2A. The red arrow points to the onset of the G1/S progression defect in 3‐MB‐PP1‐treated cells. To better visualize the G1/S delay in the presence of the inhibitor, the 24‐h time point is also shown. n = 3 replicates; representative result is shown.
  • C
    Quantification of cells (%) in individual cell cycle phases based on flow cytometry profiles of the representative replicate in Fig 2B.
  • D
    CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum‐starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from three replicates is shown.
  • E
    Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum‐containing medium. 3‐MB‐PP1 was either added or not at indicated time points after the release. Propidium iodide‐ or BrdU‐stained DNA content was measured by flow cytometry at 16 h after the release. Note, that for the BrdU staining the 3‐MB‐PP1 was added only at the time of the release (0 h) and 3, 4, 5, and 6 h after the release.
  • F, G
    Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (F)‐ and BrdU (G)‐labeled cells upon addition of 3‐MB‐PP1 at indicated time points after serum addition in the experiment depicted in Fig 2E. CTRL in Fig 2G = control sample without 3‐MB‐PP1. n = 3 replicates, representative result is shown.
  • H
    Short‐term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 μM camptothecin. A representative Western blot from three replicates is shown. FUS is a loading control.

Source data are available online for this figure.