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. 2024 May 3;144(2):156–170. doi: 10.1182/blood.2023021985

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© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure 4. — Kinase-inactivated CDK6 balances quiescent and activated transcriptional programs of HSCs. (A) Experimental workflow of low-input RNA-seq of engrafted CD45.2⁺ HSC/MPP1 cells after 2 serial rounds of transplantation. (B) Venn diagrams showing genes uniquely or commonly upregulated (left) and downregulated (right) in Cdk6^–/– and Cdk6^KM/KM compared with Cdk6^+/+ HSC/MPP1 cells after 2 serial rounds of transplantation (n=3; |Log₂FC| ≥ 0.3; adjusted P value < .2). (C-D) Gene set enrichment analysis to test for the enrichment of quiescent or activated stem cell gene sets in differentially expressed genes coming from 3 analyses: HSC/MPP1 cells of Cdk6^KM/KM compared with Cdk6^+/+ cells, Cdk6^KM/KM compared with Cdk6^–/–, or Cdk6^–/– compared with Cdk6^+/+ after 2 serial rounds of transplantation. (E-F) Transcription factor motif enrichment analysis of genes within the activated stem cell gene set that are either upregulated in (E) Cdk6^KM/KM compared with Cdk6^+/+ HSC/MPP1 cells or (F) Cdk6^–/– compared with Cdk6^+/+ HSC/MPP1 cells upon 2 serial rounds of transplantation. (G) Subcellular fractionation of Cdk6^+/+, Cdk6^–/–, and Cdk6^KM/KM HPC^LSK cells, followed by western blot analysis of CDK6. Lamin B1/regulator of chromosome condensation 1 (RCC1) served as a nuclear marker, whereas heat shock protein 90 (HSP-90) served as a cytoplasmic marker. (H) Anti-NFY-A co-immunoprecipitation (co-IP) from Cdk6^+/+, Cdk6^–/–, and Cdk6^KM/KM HPC^LSK cell extracts followed by NFY-A and CDK6 immunoblotting. IN indicates the input lysate and SN indicates the supernatant after IP. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control.

Figure 4. — Kinase-inactivated CDK6 balances quiescent and activated transcriptional programs of HSCs. (A) Experimental workflow of low-input RNA-seq of engrafted CD45.2⁺ HSC/MPP1 cells after 2 serial rounds of transplantation. (B) Venn diagrams showing genes uniquely or commonly upregulated (left) and downregulated (right) in Cdk6^–/– and Cdk6^KM/KM compared with Cdk6^+/+ HSC/MPP1 cells after 2 serial rounds of transplantation (n=3; |Log₂FC| ≥ 0.3; adjusted P value < .2). (C-D) Gene set enrichment analysis to test for the enrichment of quiescent or activated stem cell gene sets in differentially expressed genes coming from 3 analyses: HSC/MPP1 cells of Cdk6^KM/KM compared with Cdk6^+/+ cells, Cdk6^KM/KM compared with Cdk6^–/–, or Cdk6^–/– compared with Cdk6^+/+ after 2 serial rounds of transplantation. (E-F) Transcription factor motif enrichment analysis of genes within the activated stem cell gene set that are either upregulated in (E) Cdk6^KM/KM compared with Cdk6^+/+ HSC/MPP1 cells or (F) Cdk6^–/– compared with Cdk6^+/+ HSC/MPP1 cells upon 2 serial rounds of transplantation. (G) Subcellular fractionation of Cdk6^+/+, Cdk6^–/–, and Cdk6^KM/KM HPC^LSK cells, followed by western blot analysis of CDK6. Lamin B1/regulator of chromosome condensation 1 (RCC1) served as a nuclear marker, whereas heat shock protein 90 (HSP-90) served as a cytoplasmic marker. (H) Anti-NFY-A co-immunoprecipitation (co-IP) from Cdk6^+/+, Cdk6^–/–, and Cdk6^KM/KM HPC^LSK cell extracts followed by NFY-A and CDK6 immunoblotting. IN indicates the input lysate and SN indicates the supernatant after IP. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control.