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. 2023 May 25;194(1):53–69. doi: 10.1093/toxsci/kfad049

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© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 2. — Kinetics and consequences of CDK2 or CDK5 destruction using in vitro models of the bone marrow and small intestine. A and B, Kinetics of CDK2 or CDK5 destruction after dTAG-13 administration in duodenum organoids derived from HOM CDK2^dTAG or CDK5^dTAG mice. HOM CDK2^dTAG (A) and HOM CDK5^dTAG (B) duodenum organoids were treated as indicated and then the HiBit assay was performed to determine the amount of CDK2 (A) or CDK5 (B) protein. Values shown are normalized to DMSO treatment and are averages ± standard error of the mean (SEM) for 3 biological replicates. C, Degradation of CDK2 or CDK5 in CDK2^dTAG or CDK5^dTAG duodenum organoids does not affect viability. Duodenum organoids derived from WT, CDK2^dTAG or CDK5^dTAG HOM mice were treated with dTAG-13 for 4 days, then viability was measured by CellTiter-Glo 3D. Values shown are normalized to DMSO treatment and are averages ± SEM for 3 biological replicates. D and E, No significant changes in gene transcription after degrading CDK2 in CDK2^dTAG duodenum organoids. Duodenum organoids derived from HOM CDK2^dTAG mice were treated for 6 h (D) or 24 h (E) with 250 nM dTAG-13 or DMSO, QuantSeq was performed, and differential gene expression analysis was performed between the 2 groups. n = 3 biological replicates. FDR, false discovery rate. F and G, No significant changes in gene transcription after degrading CDK5 in CDK5^dTAG duodenum organoids. Duodenum organoids derived from HOM CDK5^dTAG mice were treated for 6 h (F) or 24 h (G) with 250 nM dTAG-13 or DMSO, QuantSeq was performed, and differential gene expression analysis was performed between the 2 groups. n = 3 biological replicates. (H and I) No significant changes in gene transcription after CDK2 KO in CDK2 cKO duodenum organoids. Duodenum organoids derived from HOM CDK2 cKO mice were treated for 24 h (H) or 48 h (I) with 1 µM 4-hydroxytamoxifen (4-OHT) or DMSO, QuantSeq was performed, and differential gene expression analysis was performed between the 2 groups. n = 3 biological replicates. J, Potent degradation of CDK2 or CDK5 in BMDCs from CDK2^dTAG and CDK5^dTAG mice. Bone marrow cells were derived from multiple CDK2^dTAG or CDK5^dTAG HOM mice, pooled, treated with dTAG-13 in vitro for 6 h at the indicated concentrations, and the HiBit assay was performed to determine levels of CDK2 or CDK5 protein. Values are normalized to DMSO treatment and are averages ± SEM for 3 technical replicates. K, No change in viability after degrading CDK2 or CDK5 in BMDCs from CDK2^dTAG or CDK5^dTAG mice. Bone marrow cells were derived from 3–4 WT, CDK2^dTAG or CDK5^dTAG HOM mice, pooled, treated with dTAG-13 in vitro for 4 days at the indicated concentrations, and cell viability measured via CellTiter-Glo. Values are normalized to DMSO treatment and are averages ± SEM for 3 technical replicates. L, No significant changes in gene transcription after degrading CDK2 in BMDCs from CDK2^dTAG mice. BMDCs from HOM CDK2^dTAG were treated with 250 nM dTAG-13 or DMSO for 24 h, QuantSeq was performed, and differential gene expression analysis was performed between the 2 groups. n = 3 technical replicates. BMDCs, bone marrow derived cells.