Fig 1: PCGF1 restricts self-renewal of granulocyte-macrophage progenitors (GMPs) by attenuating Hoxa9 expression.(A) Growth of control and Pcgf1?/? GMPs in culture. Cells were cultured in triplicate under myeloid culture condition-2 (25 ng/mL SCF, TPO, Flt3L, and IL-11 and 10 ng/mL IL-3 and GM-CSF). Data are shown as the mean ± SD (n = 3). (B) Growth of control and Pcgf1?/? hematopoietic stem cells (HSCs) under myeloid culture condition-2 (25 ng/mL SCF, TPO, Flt3L, and IL-11 and 10 ng/mL IL-3 and GM-CSF). Cells were cultured in triplicate. The proportion of GMPs in culture is depicted on the right panel. Data are shown as the mean ± SD (n = 3). (C) Replating assay data. 700 LSK cells were plated in a methylcellulose medium containing 20 ng/mL of SCF, TPO, IL-3, and GM-CSF. After 10 d of culture, colonies were counted and pooled, and 1 × 104 cells were then replated in the same medium every 7 d. Data are shown as the mean ± SEM (n = 3). (D) Proportion of immunophenotypic GMPs with nuclear ß-catenin in control and Pcgf1?/? immunophenotypic GMPs in HSC culture on day 16 in (B). Representative immunofluorescent signals of ß-catenin in control immunophenotypic GMPs are shown on the right panel. Data are shown as the mean ± SEM (n = 3). (E) Quantitative RT-PCR analysis of Hoxa9, Irf8, Csf1r, and Il-6ra in sorted control and Pcgf1?/? immunophenotypic GMPs in HSC culture in (B) at the indicated time points. Hprt1 was used to normalize the amount of input RNA. Data are shown as the mean ± SEM (n = 3). (F) Gene set enrichment analysis (GSEA) using RNA-seq data. The gene sets used are indicated in Supplementary file 1. (G) Growth of mock control and Hoxa9-expressing LSK cells. LSK cells transduced with a Hoxa9 retrovirus harboring mCherry marker gene were cultured in triplicate under myeloid culture condition-2 (25 ng/mL SCF, TPO, Flt3L, and IL-11 and 10 ng/mL IL-3 and GM-CSF). The proportion of GMPs in culture is depicted on the right panel. Data are shown as the mean ± SD (n = 4). (H) Proportion of GMPs with nuclear ß-catenin in mock control and Hoxa9-expressing GMPs in LSK culture on day 12 in (G). Data are shown as the mean ± SEM (n = 5–6). (I) Model of the molecular network controlling GMP self-renewal and differentiation. *p<0.05; **p<0.01; ***p<0.001 by the Student’s t-test (A–D, H, and G) or the one-way ANOVA (E). Each symbol is derived from an individual culture. Figure 5—source data 1.Raw data for Figure 5.