Fig 2: Hypothetical model for the epigenetic role of Asxl1 in regulating Nmyc expression during mouse fetal lung development.Details are provided in the Discussion section

Fig 3: Interaction between ASXL1 and HCF-1.a Affinity purification of the ASXL1-bound complex. HEK293 cells stably expressing Flag-tagged murine Asxl1 (F-Asxl1) were subjected to IP using anti-Flag antibody-coupled beads. Affinity-purified ASXL1 complex proteins were separated by SDS–PAGE and stained with Coomassie Blue. Proteins identified by LC–MS/MS are indicated at right. b, c Endogenous interaction between ASXL1 and HCF-1 in H1299 cells. Reciprocal IP using an anti-ASXL1 antibody (b) and anti-HCF-1 antibody (c) was followed by WB using the indicated antibodies. d Mapping of the HCF-1 domain responsible for ASXL1 binding. HEK293T cells were transfected with plasmids expressing Flag-Asxl1 and Myc-tagged truncation mutants of HCF-1 (aa1–434, 435–1011, and C’, aa1436–2035). IP using an anti-Flag antibody was followed by WB using an anti-Myc antibody. Asterisk indicates IgG heavy chain. e Interaction between ASXL1 and HCF-1 in vitro. GST pull-down assay was performed using GST-Asxl1 (aa1193–1514) and His-tagged HCF-1N (aa1–434) protein produced by in vitro translation. A Coomassie Blue-stained gel is shown

Fig 4: Asxl1 represses Nmyc expression.For quantification, three independent experiments were performed. Data are means ± SD (**P < 0.01, ***P < 0.001). a Effect of E2F1 on Nmyc promoter activation. The WT (D6 WT) and mutant (D6 MT) E2F1-responsive sites in the Nmyc promoter are indicated. HEK293T cells were transfected with an Nmyc promoter-driven luciferase reporter gene and E2F1 expression vector (0, 0.01, 0.05, or 0.1 µg). b E2F1 binding to the E2F1-responsive site in the Nmyc promoter. Schematic depiction of primer pairs to map the E2F1-binding site (shown by yellow). ChIP assay using an anti-Flag antibody and primer set for the human Nmyc promoter in H460 and A549 cells transfected with Flag-E2F1. Chromatin binding is shown as a percentage of the input. c Effect of Asxl1 on Nmyc promoter repression. HEK293T cells were transfected with the WT or mutant Nmyc luciferase reporter gene and ASXL1 (0.2, 0.4, 0.6, and 0.8 µg). d ASXL1 binding to the Nmyc promoter. ChIP-qPCR was performed using an anti-ASXL1 antibody and primer set for Nmyc promoter in A549 and H460 cells

Fig 5: Effect of Asxl1 on the proliferation of lung epithelial cells.a Cell proliferation was determined by immunostaining with Ki-67 and PCNA at E18.5. Scale bar, 100 µm. b mRNA expression levels were measured by RT-qPCR. c Differentiation was assayed using antibodies against the lung epithelial marker Ccsp and the muscle marker aSma at E18.5. d mRNA expression levels are shown. Data are means ± SD (n = 6, **P < 0.01)