CDK1 and CDK2 regulate NICD1 turnover and the periodicity of the segmentation clock (2019)
Figure 7

Figure 7. Purvalanol B treatment reduces the NICD-FBXW7 interaction and CDK1 or CDK1 inhibition delays the pace of the segmentation clock.

Purvalanol B treatment reduced the NICD-FBXW7 interaction. 200 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 in combination with 0.1 µM of Purvalanol B were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin has been used as loading control.

Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.

E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of Purvalanol B (1 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin was used as loading control.

E10.5 mouse tails were bissected down the midline. One half (+) was cultured for 4 hrs in the presence of RO-3306 (10 µM). The contralateral half (-) was cultured for 4h in the presence of DMSO. Control or treated explants were pooled and NICD levels were detected by western blot. β-Actin has been used as loading control.

Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 1µM of Purvalanol B for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. Purvalanol B treated explant has one less somite than the control explant and the treated explant is in the same late phase 1 of the oscillation cycle of dynamic mLfng mRNA expression indicating it is a whole cycle delayed compared to the "-" explant. n=18. Scale bar is 100 µm.

Bissected E10.5 mouse PSM explants were cultured in the absence (-) or presence (+) of 10 µM of RO-3306 for 4 hours and then analysed by in situ hybridization for mLfng mRNA expression. RO-3306 treated explant is two phases behind in the oscillation cycle of dynamic mLfng mRNA expression indicating there is a delay in the oscillation compared to the "-" explant. n=10. Scale bar is 100 µm.

The mean log2 fold change in successive somite length at position +1 (last formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.0437 for RO-3006 and p= 0.0524 for PurB, compared to DMSO. n=14.

The mean log2 fold change in successive somite length at position +2 (penultimate formed somite) is plotted for different treatment conditions. Error bars denote standard error of the mean. Paired t-test analysis was performed at 5% significance level, with p= 0.6409 for RO-3006 and p= 0.4065 for PurB, compared to DMSO. n=14.

Table 1. CDK1 and CDK2 exhibit specific activity against a NICD phospho-peptide by in vitro kinase assay.

Three NICD phospho-peptides were tested for the activity of seven different kinases (CDK1, CDK2, CDK5, CDK7, CDK9, CK1α and GSKβ). A known substrate was used as control for each kinase. The specific activity of each kinase is expressed in U/mg.

Peptide 1 contains serine residues 2513 and 2516 (HPFLTPSPESPDQWSSSSPH).

Peptide 2 includes serine 2538 (NVSDWSEGVSSPPTSMQSQIA).

Peptide 3 encompasses serine residue 2141 (GTPTLSPPLCSPNGYLGSLKP).

Table 2. Purvalanol B and RO-3306 small molecule inhibitors delays the pace of the segmentation clock.

Summary table of number of somites and clock delays before and after treatment with Purvalanol B (A) and RO-3306 (B) inhibitors, compared to DMSO, in E10.5 mouse explants.

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