Retroviral overexpression of CRLF2 in hematopoietic progenitors results in increased proliferation and STAT-5 phosphorylation. Conversely, knockdown of CRLF2 results in decreased proliferation. (A) CFC assay on E13.5 mouse fetal liver cells infected with hCRLF2 or EV control retroviruses and grown in methylcellulose semisolid medium supplemented with IL-7, SCF, and FLT3L. Values are average of 2 independent experiments; error bars indicate SDs. All 4 platings show statistical significance between EV and hCRLF2 for number of colonies (unpaired Student t test, first and second plating P < .001, third plating P < .001); only hCFL2 colonies grew in fourth replating. EV colonies represented 44.9% ± 13.3%, 57.1% ± 11.2%, and 0.43% ± 0.3% of the number of CRLF2 colonies at first, second, and third plating, respectively. (B) Images showing relative sizes of colonies formed by hCRLF2- and EV-transduced hematopoietic progenitors at 2 platings. (C) Lysed hCRLF2-infected cells from second plating have increased STAT-5 Y694 phosphorylation compared with EV cells. (D) A higher proportion of CRLF2-overexpressing cells at second plating show less differentiated phenotype compared with EV cells. CD43⁺/B220⁺/CD19⁺ pre-B cells were more prevalent in EV than hCRLF2 cell populations (P < .001), suggesting that increased proliferation is accompanied by maintenance of a larger pool of cells with immature immunophenotype: CD43⁺/B220⁺/CD19⁻ corresponding to Hardy fraction A. (E) flow cytometric evaluation of hCRLF2 surface expression in cells from hCRLF2 hematopoietic progenitor colonies at second plating. Solid histogram represents cells stained with a negative control antibody. hCRLF2-positive cells with lower hCRLF2 expression have a higher proportion of CD43⁺/CD19⁺ cells (81%) compared with cells with high expression of hCRLF2 (23.5%). By third plating, all CRLF2 cells are CD43⁺/CD19⁺ with almost negligible μ expression (not shown). (F) Cell lysates were prepared from the MUTZ5 cell line, 3 and 4 days following transfection, with either scrambled control (SC) or CRLF2 shRNA knockdown plasmids (071 and 195) and probed with antibodies specific for CRLF2 or β-actin. (G) MUTZ5 cells were transfected with either SC or CRLF2 shRNA knockdown plasmid 071 (KD) and proliferation was monitored using an MTS assay (n = 3). The down-regulation of CRLF2 expression in MUTZ5 results in a significant decrease in proliferation (P = .029, unpaired t test).