CDK6 influences (re-)plating capacities of BCR-ABL^p210+ LSKs in vitro. (A) Experimental setup: Cdk6^+/+ and Cdk6^−/− BM cells were cocultivated on BCR-ABL^p210 producer cells for 48 hours, sorted by high-purity FACS, and either subjected to colony formation assays (CFA) (B-D) or analyzed by qPCR (E). (B) Three different cell numbers of BCR-ABL^p210+ LSKs were seeded and colony numbers were counted 8 days after coculture (technical duplicates; *P < .05). (C) All colonies were harvested and reseeded to a second round of replating. Colonies were counted after 8 days (technical duplicates; *P < .05). (D) After each round, colonies were harvested and analyzed by FACS for the presence of remaining BCR-ABL^p210+ LSKs (*P < .05). (E) BCR-ABL^p210+ LSKs were sorted by FACS and Egr1 expression was analyzed by qPCR (BM cells of 3 individual mice per genotype were pooled; qPCR was performed in technical triplicates; *P < .05). (F) Knockdown constructs Egr1 #1 and Egr1 #2 or a control vector targeting Renilla were introduced into Cdk6^−/− BCR-ABL^p210+ LSKs and subjected to colony formation. Colonies were again counted 8 days after seeding (****P < .0001). (G) Representative pictures of colonies on day 8 (magnification: ×4).