cdc7 kinase inhibitors  (MultiTarget Pharmaceuticals)

Journal: bioRxiv

Article Title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

doi: 10.1101/2021.11.02.466939

Figure Lengend Snippet: A) Ewing Sarcoma cells (A673, RD-ES and SK-ES-1) and the non-Ewing, osteosarcoma cell line U2OS were treated with increasing concentrations of the DDK inhibitors XL413 and TAK-931 for 72 hours and cell viability was measured using CCK-8 cell viability reagent. Relative viability was calculated based on DMSO treated cells (n=3 technical replicates). 2-way ANOVA multiple comparisons, ***p<0.001, ****p<0.0001.B) Cells were treated with either 0.1% DMSO, 5µM XL413 (left panel) or 1µM TAK-931 (right panel) for 48 hours. Whole cell lysates were collected, and western blot was performed for the specified proteins. GAPDH was used as a loading control (representative image of 3 independent experiments).C) Ewing Sarcoma cells were treated with 0.1% DMSO, 5µM XL413 or 1µM TAK-931 for 48 hours. Cells were stained with anti-Annexin-V (AlexaFluor-488 conjugated) and propidium iodide and analyzed using flow cytometry. Apoptotic cells were designated as Annexin-V (upper right and lower right quadrants) positive cells and were measured using FCS express v7 (n=3 biological replicates). Unpaired t test, ***p<0.001, **p<0.01.

Article Snippet: The CDC7 kinase inhibitor, XL413, was purchased from Sigma-Aldrich (cat. No. SML1401; batch #: 0000036390) and dissolved in sterile filtered ddH 2 O to a final concentration of 5mM.

Techniques: CCK-8 Assay, Western Blot, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

doi: 10.1101/2021.11.02.466939

Figure Lengend Snippet: A) Ewing Sarcoma cells and U2OS cells were treated with either 0.1% DMSO (black line), 300nM TAK-931 (red line) or 5µM XL413 (blue line) for 8 hours. Then, in the presence of DDKi, cells were incubated with 10µM EdU for 30 minutes. Cells were then fixed and stained for EdU (AlexaFluor488) (X-axis) and EdU intensity was analyzed using flow cytometry. Representative image of three independent experiments. B & C) Ewing Sarcoma cells and U2OS cells were treated with 0.1% DMSO (72 hours), 300nM TAK-931 or 1µM XL413 for 24, 48 and 72 hours, DNA was stained with propidium iodide and DNA content was analyzed using flow cytometry. Cell cycle distribution was measured using FCS Express v7. B) Representative histograms of three independent experiments showing only the results obtained with A673 and U2OS cells. C) Quantification of cell cycle distribution; G1 = grey bars, S = white bars, G2/M = blue bars, sub-G1 = red bars (n=3 biological replicates).

Article Snippet: The CDC7 kinase inhibitor, XL413, was purchased from Sigma-Aldrich (cat. No. SML1401; batch #: 0000036390) and dissolved in sterile filtered ddH 2 O to a final concentration of 5mM.

Techniques: Incubation, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

doi: 10.1101/2021.11.02.466939

Figure Lengend Snippet: Ewing Sarcoma cells and U2OS cells were treated with 0.1% DMSO (72 hours), 300nM TAK-931 or 1µM XL413 for 24, 48 and 72 hours, DNA was stained with propidium iodide and DNA content was analyzed using flow cytometry. Cell cycle distribution was measured using FCS Express v7. A) Representative histograms of three independent experiments B) Quantification of cell cycle distribution; G1 = grey bars, S = white bars, G2/M = blue bars, sub-G1 = red bars (n=3 biological replicates).

Article Snippet: The CDC7 kinase inhibitor, XL413, was purchased from Sigma-Aldrich (cat. No. SML1401; batch #: 0000036390) and dissolved in sterile filtered ddH 2 O to a final concentration of 5mM.

Techniques: Staining, Flow Cytometry

Journal: bioRxiv

Article Title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

doi: 10.1101/2021.11.02.466939

Figure Lengend Snippet: A) Representative images of mitotic abnormalities induced in A673 cells upon DDKi treatment. a) anaphase bridges, b) lagging chromosomes, c) four centrosomes, d) three centrosomes, e) mitotic catastrophe, f) unclassified mitotic abnormality (representative images of two independent experiments). B) A673 cells were treated with 0.1% DMSO (72 hours), 300nM TAK-931 or 1µM XL413 for 24, 48 and 72 hours and DNA content was stained with DAPI. Percent cells with micronuclei were quantified as any cell that displayed 1 or more micronuclei (white arrows) structure around the nucleus (n = 3 biological replicates). 2way ANOVA, ****p<0.0001, **p<0.01. C) A673 cells were treated with either 0.1% DMSO, 300nM TAK-931 or 1µM XL413 for 24, 48 and 72 hours. Cells were fixed and stained for yH2AX (AlexaFluor 488 – green) and DNA (DAPI – blue). D) Corrected total cellular fluorescence intensity for yH2AX was calculated using FIJI image analysis software (representative images of two biological replicates ∼100 cells were quantified per condition). 2way ANOVA, *p<0.05, ****p<0.0001.

Article Snippet: The CDC7 kinase inhibitor, XL413, was purchased from Sigma-Aldrich (cat. No. SML1401; batch #: 0000036390) and dissolved in sterile filtered ddH 2 O to a final concentration of 5mM.

Techniques: Staining, Fluorescence, Software

Journal: bioRxiv

Article Title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

doi: 10.1101/2021.11.02.466939

Figure Lengend Snippet: Ewing Sarcoma cells and U2OS cells were treated with 0.1% DMSO (72 hours), 300nM TAK-931 or 1µM XL413 for 24, 48 and 72 hours, DNA was stained with propidium iodide and DNA content was analyzed using flow cytometry. Percentages of cells with <2N (black dotted line) and >4N (blue dotted line) was measured using FSC Express v7 (n=3 biological replicates) Ordinary one-way ANOVA, *p<0.05, ***p<0.001, ****p<0.0001.

Article Snippet: The CDC7 kinase inhibitor, XL413, was purchased from Sigma-Aldrich (cat. No. SML1401; batch #: 0000036390) and dissolved in sterile filtered ddH 2 O to a final concentration of 5mM.

Techniques: Staining, Flow Cytometry

Journal: bioRxiv

Article Title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

doi: 10.1101/2021.11.02.466939

Figure Lengend Snippet: A &B) Ewing sarcoma cells were treated with 300nM TAK-931 or 1µM XL413 +/- 20µM DRB for 24 hours. DNA content was analyzed using flow cytometry. A) Representative histogram images of A673 cells of 2 independent experiments. B) Cell cycle quantification of A673 (top), RD-ES (middle) and SK-ES-1 (bottom) under the specified conditions (n=2 biological replicates).

Article Snippet: The CDC7 kinase inhibitor, XL413, was purchased from Sigma-Aldrich (cat. No. SML1401; batch #: 0000036390) and dissolved in sterile filtered ddH 2 O to a final concentration of 5mM.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: DDK inhibition disrupts replication leading to mitotic catastrophe in Ewing sarcoma

doi: 10.1101/2021.11.02.466939

Figure Lengend Snippet: A) TC32 cells were treated with specified concentrations of DDKi - XL413 (left-grey bars) and TAK-931 (right – black bars) for 72 hours and cell viability was assessed using CCK8. Relative viability was calculated based on DMSO treated controls (n = 3 technical triplicates; Ordinary one-way ANOVA compared to DMSO treated control ****p<0.0001). B) TC32 cells were treated with either 0.1% DMSO, 5µM XL413 or 1µM TAK-931 for 48 hours. Protein lysate was collected, and a western blot was run for specific proteins. GAPDH was used as a loading control (n = 1). C) TC32 cells were treated with 300nM TAK-931 for specified times (DMSO = 0.1% DMSO for 72 hours). DNA content histograms were generated using flow cytometry and cell cycle distributions were quantified in panel D. E) TC32 cells were treated with either 0.1% DMSO, 1µM Nocodazole (24 hours) or 300nM TAK931 for 24 or 48 hours. Protein lysate was collected, and a western blot was run for specific proteins. GAPDH was used as a loading control (n = 1). Upon DDK inhibition, TC32 cells (TP53 WT) experience a similar reduction in cell viability, pattern of cell cycle delay and apoptosis asTP53 mutant cells. Importantly, these cells, too, do not experience a mitotic accumulation, suggesting that the lack of mitotic delay upon DDK inhibition in A673 cells is not due to the lack of p53 function.

Article Snippet: The CDC7 kinase inhibitor, XL413, was purchased from Sigma-Aldrich (cat. No. SML1401; batch #: 0000036390) and dissolved in sterile filtered ddH 2 O to a final concentration of 5mM.

Techniques: Western Blot, Generated, Flow Cytometry, Inhibition, Mutagenesis