Impaired activation of dormant Cdk6^−/− HSCs after poly(I:C) treatment. (A) Cdk6^+/+ and Cdk6^−/− mice received a pulse of BrdU i.p. (1 mg/mouse) that was followed by a 10-day period of BrdU administration via drinking water (1 mg/mL) (n = 8 per genotype). After a 70-day chase period, the mice cohort was split and BrdU⁺ fraction A cells were analyzed with (n = 4 per genotype) or without (n = 4 per genotype) a 24-hour preceding injection of poly(I:C) (10 µg/g body weight). Treatment with poly(I:C) resulted in a 10-fold reduction of BrdU⁺ fraction A cells in Cdk6^+/+, but only in a 2.5-fold reduction in Cdk6^−/− mice. (B) Cdk6 and Cdk4 mRNA expression levels in fraction A cells of Cdk6^+/+ and Cdk6^−/− mice that had received poly(I:C) or PBS are shown (n = 3 for each genotype; qPCR analyses were performed in triplicate; **P < .01). (C) Egr1 mRNA expression levels in fraction A cells of Cdk6^+/+ and Cdk6^−/− mice that had received poly(I:C) or PBS are shown (n = 6 for each genotype; qPCR analyses were performed in triplicate; ***P < .001). (D-E) ChIP assays were performed in an HPC-7 hematopoietic progenitor cell line (D) and in primary Cdk6^+/+ LSKs (E). Protein–DNA complexes were immunoprecipitated using antibodies directed against CDK6 and analyzed by qPCR for their presence on the Egr1 promoter region. Vegf-A and/or p16^INK4a promoter regions were used as positive controls. Bar graphs depict fold enrichment over a negative region downstream of CD19. (F) Cdk6^−/− LSKs were sorted and coincubated with Cdk6/GFP⁺, Cdk6^K43M/GFP⁺, or empty/GFP⁺ GP⁺ producer cells (n = 3 per genotype). After 48 hours, GFP⁺ cells were high-purity sorted by FACS and analyzed by qPCR. Bar graphs depict Egr1 mRNA expression levels (technical triplicates; *P < .05, **P < .01).