![CDK6 shapes the HSC transcriptomic landscape in a kinase-inactivated, kinase-dependent, and -independent manner. (A) Flow cytometry analysis of isolated BM from Cdk6+/+, Cdk6–/–, and Cdk6KM/KM mice. Cell numbers of HSCs (LSK [Lin–Sca-1+c-kit+] CD34–CD48–CD150+CD135–), MPP1 (LSK CD34+CD48–CD150+CD135–), MPP2 (LSK CD48+CD150+), and MPP 3/4 (LSK CD48+CD150–) (n = 10; mean ± standard error of the mean [SEM]). (B) Experimental scheme of 10x Genomics scRNA-seq including flow cytometry sorting of LSK cells of Cdk6+/+, Cdk6–/–, and Cdk6KM/KM BM (top). Uniform manifold approximation and projection (UMAP) visualization of 11 LSK cell clusters (bottom). Colors indicate different clusters. (C) UMAP of 9 HSPC subclusters with color code. (D) Bar chart of HSPC subcluster size differences of either Cdk6–/– or Cdk6KM/KM compared with the Cdk6+/+ control (Log2FC of percent cluster sizes relative to Cdk6+/+). (E) UMAP of Cdk6+/+, Cdk6–/–, and Cdk6KM/KM HSPC cluster. The arrow indicates HSC subcluster. (F) Classification of CDK6 states: kinase-inactivated, kinase-dependent, and loss of CDK6 (top). Venn diagrams showing number of genes of the HSC subcluster uniquely (bottom) or commonly upregulated (left) and downregulated (right) in Cdk6KM/KM and Cdk6–/– compared with Cdk6+/+ (|Log2FC| ≥ 0.3). (G) UMAP showing Cdk6+/+, Cdk6–/–, and Cdk6KM/KM HSPCs overlaid with the HSC–associated proliferation gene signature (Psig).16 The 15% of cells with the lowest Psig score (compare methods) are indicated in blue. Violin plots depict Psig and HSC–associated quiescent signature (Qsig) of all 3 genotypes. Cycle, cell cycle; Ery, erythroid; Rep, replication. ∗P < .05.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/144/2/10.1182_blood.2023021985/2/blood_bld-2023-021985-gr1.jpeg?Expires=1750119853&Signature=MMuPDqPhcUpK0R56xQKobz4~AP9nji-jDUEeTRT-Z-aqbbUHhtN7slzu-vTxMkClwPGkYbajKfZTOjbqG0IgZJBB5OS5FKv3RcjLk6Ts0n9w38olAle6UCf1GRYw8G091fV-tqzSoKknbQPqG4rxJZYK~A1vaAK3AsMdBuhx1iqVOFc0DBhu0~X8meF2UPqpL3QQmuA5fOd25Pt5JWTqdv6R9RhBSP7lJ1wNPQmxewqI2yRrWrcdCWNcILg5K0CC2EaEl6s3WxnQlgj5THIAhCv43NwEg9D~m24BPXZpEuAdQeteIINCych27ThUUnGoivDvUxWvyT8tVLbkehKD~g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CDK6 shapes the HSC transcriptomic landscape in a kinase-inactivated, kinase-dependent, and -independent manner. (A) Flow cytometry analysis of isolated BM from Cdk6+/+, Cdk6–/–, and Cdk6KM/KM mice. Cell numbers of HSCs (LSK [Lin–Sca-1+c-kit+] CD34–CD48–CD150+CD135–), MPP1 (LSK CD34+CD48–CD150+CD135–), MPP2 (LSK CD48+CD150+), and MPP 3/4 (LSK CD48+CD150–) (n = 10; mean ± standard error of the mean [SEM]). (B) Experimental scheme of 10x Genomics scRNA-seq including flow cytometry sorting of LSK cells of Cdk6+/+, Cdk6–/–, and Cdk6KM/KM BM (top). Uniform manifold approximation and projection (UMAP) visualization of 11 LSK cell clusters (bottom). Colors indicate different clusters. (C) UMAP of 9 HSPC subclusters with color code. (D) Bar chart of HSPC subcluster size differences of either Cdk6–/– or Cdk6KM/KM compared with the Cdk6+/+ control (Log2FC of percent cluster sizes relative to Cdk6+/+). (E) UMAP of Cdk6+/+, Cdk6–/–, and Cdk6KM/KM HSPC cluster. The arrow indicates HSC subcluster. (F) Classification of CDK6 states: kinase-inactivated, kinase-dependent, and loss of CDK6 (top). Venn diagrams showing number of genes of the HSC subcluster uniquely (bottom) or commonly upregulated (left) and downregulated (right) in Cdk6KM/KM and Cdk6–/– compared with Cdk6+/+ (|Log2FC| ≥ 0.3). (G) UMAP showing Cdk6+/+, Cdk6–/–, and Cdk6KM/KM HSPCs overlaid with the HSC–associated proliferation gene signature (Psig).16 The 15% of cells with the lowest Psig score (compare methods) are indicated in blue. Violin plots depict Psig and HSC–associated quiescent signature (Qsig) of all 3 genotypes. Cycle, cell cycle; Ery, erythroid; Rep, replication. ∗P < .05.
