Fig. 5: Meriolins and other known CDK inhibitors induce cell cycle arrest and reduce proliferation in sublethal doses.

A The detection of cell cycle phases was performed by flow-cytometric analysis of the DNA content of propidium iodide-stained nuclei using the Nicoletti assay [41]. Since the DNA content doubles during S phase, a higher fluorescence intensity can be detected in G₂ compared to G₁ phase. Due to the caspase dependent DNA fragmentation and subsequent leakage of fragmented DNA from apoptotic nuclei, apoptosis can be determined by the formation of hypodiploid nuclei (HN). Cell cycle analysis in Ramos cells was determined after 24 h treatment without (left diagram) and with (right diagram) pre-and co-treatment of the pan-caspase inhibitor QVD (10 µM), followed by a sublethal dosage of each of the respective CDK inhibitor (meriolin 16 (0.02 µM), meriolin 36 (2 µM), R547 (1 µM), flavopiridol (0.1 µM), roscovitine (10 µM), SNS-032 (0.15 µM), meriolin 3 (0.1 µM), dinaciclib (0.01 µM), zotiraciclib (0.1 µM)). The broad kinase inhibitor and potent apoptotic stimulus staurosporine (STS; 2.5 µM) was used as positive control for apoptosis induction and DMSO (0.1% v/v) as diluent control. Untreated cells are shown as control. Error bars = Mean ± SD of three independent experiments performed in triplicates. B Proliferation was measured by the incorporation of BrdU with the BrdU cell proliferation assay. 1 × 10⁴ Ramos cells were pre-incubated with the compounds for 24 h: meriolin 16 (0.02 µM), meriolin 36 (0.1 µM), meriolin 3 (0.1 µM), dinaciclib (0.01 µM), R547 (1 µM), or medium (Control). One hour post treatment with the compounds of interest, the BrdU solution was added and further incubated with the compounds in order to monitor residual proliferative capacity. Error bars = Mean ± SD of ≥4 independent experiments performed in triplicates. C Measurement of proliferation-inhibition by microscopic analysis of EdU-incorporation in HeLa cells. In the EdU assay, the incorporation of EdU (thymidine nucleoside analog) into the DNA was measured and serves as a parameter of proliferation. HeLa cells were treated with the respective IC₂₅ values (as evaluated in E) of meriolin 16 (0.04 µM), meriolin 36 (0.4 µM) or DMSO (0.1% v/v) as solvent control. The EdU-incorporation was analyzed by immunofluorescence and exemplary microscopy images are shown (green: EdU-incorporation; blue: DAPI stained nuclei). D The quantification of EdU-positive cells from several microscopy images is shown as bar graph of relative EdU positive cells in %. (Biological replicates n = 1–2; 10 cells were counted for DMSO, 30 for meriolin 16, 22 for meriolin 36). E Evaluation of IC₂₅ and IC₅₀ values of HeLa cells. HeLa cells were treated for 24 h with concentrations between 0.01 μM and 30 μM of meriolin 16 or meriolin 36. Cell viability was measured by AlamarBlue assay; n = 1.