Fig 2: Reticular cell subset differentiation during splenic white pulp development. a, b Time course analysis of EYFP-expressing cells in spleens from Ccl19-iEYFP mice harvested at the indicated stages. Confocal microscopic analysis depicting αSMA+ myofibroblasts (a) and PDPN+ TRC (b). Boxes in (b) indicate magnified regions in the lower row. Microscopy data are representative for two or more independent experiments (n ≥ 4 mice per group). c, d Flow cytometric analysis of CD45−TER119−EYFP+ cells using antibodies against PDPN, CD157 and Sca-1. Values from one representative experiment indicate percentage of the respective population shown in (c) and cumulated values for proportions of reticular cell subsets shown in (d) (n > 5 mice per group from two to three independent experiments, mean ± SEM). e, f Assessment of cellular proliferation using Ki67 staining in spleens of Ccl19-iEYFP mice at the indicated age with representative section in (e) and quantification of the proportion of Ki67 expression in EYFP+ cells (f) (n > 4 mice per group from three independent experiments, mean ± SEM). Statistical analysis was performed using a one-way ANOVA with Tukey’s post test. Source data are provided as Source Data file

Fig 3: FoxM1 deficiency impairs SC maintenance by impeding cell cycling of SCs.a Skeletal muscles were harvested from mice at 8 months of age and then were digested as mononuclear cells. The cells were stained with cell surface markers (CD45, Sca1, CD11b, CD31,CD34, a7-integrin). The population of SCs (defined as CD45-Sca1-CD11b-CD31-CD34+a7-integrin+ cells) was analyzed by flow cytometry. b Quantification of the relative percentage of SCs per total mononuclear cells isolated from the skeletal muscles of mice at 8 months of age (n = 4). c Skeletal muscles were harvested from mice (8 months of age) and digested as myofibers. Representative fluorescent images of fresh myofibers immunostained with Pax7 antibody (green) and DAPI (blue). Scale bars, 50 µm. d Quantification of the numbers of Pax7+ SCs per myofiber of mice at 8 months of age (n = 5, 20 myofibers per mouse). e The mononuclear cells were isolated from the skeletal muscles of mice (2 months of age) and stained with cell surface markers, DAPI and Ki67. The quiescent percentage of SCs was analyzed by flow cytometry. f Quantification of the percentage of G0 phase of SCs in mice (2 months of age) (n = 7). g The mononuclear cells were isolated from the skeletal muscles of mice (2 months of age) and were stained with cell surface markers and DAPI. The cell cycle of SCs was analyzed by flow cytometry. h Quantification of the percentage of cell cycle distribution of SCs in mice at 2 months of age (n = 4). i In vitro proliferation assay (CCK8) of cultured SCs isolated from mice (2 months of age; n = 5). j The mononuclear cells were isolated from the skeletal muscles of mice (2 months of age) and were stained with SC markers, as well as Annexin-V and DAPI. The percentage of apoptotic SCs (Annexin-V+ SCs) was analyzed by flow cytometry (n = 4). Error bars represent the means ± SD. **p < 0.01, ***p < 0.001; Student’s t test.

Fig 4: PCGF1 negatively regulates granulocyte-macrophage progenitor (GMP) self-renewal.(A) Immunofluorescence staining of bone marrow (BM) sections from control and Pcgf1?/? mice. Magnified images are depicted in the middle panels. Dotted lines denote clusters of GMP (Lin–Sca-1–CD41–IL-7Ra–c-Kit+Fc?R+) (c-Kit, red; Fc?R, blue; merged, purple). The right panel shows c-Kit+Fc?R+ purple cell area in BM sections relative to that in control. Data are shown as the mean ± SEM (n = 3). (B) Strategy to analyze emergency myelopoiesis induced by one shot of 5-FU (150 mg/kg). (C) Absolute numbers of total BM cells, LSK cells, GMPs, and Mac-1+ myeloid cells in a unilateral pair of femur and tibia and Mac-1+ myeloid cells in peripheral blood (PB) at the indicated time points post-5-FU injection. Data are shown as the mean ± SEM (n = 4;* versus day 0 for each genotype). Circles represent the data from individual mice. (D) A Venn diagram showing the overlap between upregulated differentially expressed genes (DEGs) in 5-FU treated GMPs on day 8 and upregulated DEGs in Pcgf1?/? GMPs. (E) Quantitative RT-PCR analysis of Hoxa9 in LSK cells and GMPs. Hprt1 was used to normalize the amount of input RNA. Data are shown as the mean ± SEM (n = 3). (F) Chromatin immunoprecipitation (ChIP) qPCR assays for H2AK119ub1 at the Hoxa9 locus in GMPs from WT mice on days 0, 10, and 14 post-5-FU treatment. The relative amounts of immunoprecipitated DNA are depicted as a percentage of input DNA. Data are shown as the mean ± SEM (n = 3). (G) Fold changes in H2AK119ub1 levels in Pcgf1?/? GMPs relative to control GMPs at the promoters of non-DEGs and DEGs up- and downregulated in day 8 GMPs compared to day 0 GMPs post-5-FU treatment. (H) UMAP plots illustrating the identification of cell clusters based on single-cell transcriptomic profiling of control and Pcgf1?/? myeloid progenitors (Lin–Sca-1–c-Kit+). (I) Principal component analysis (PCA) plots of control and Pcgf1?/? GMPs individually and in combination. (J) UMAP and violin plots showing expression of Hoxa9, Irf8, Csf1r, and Il-6ra in control and Pcgf1?/? myeloid progenitors. (K) Gene Ontology and pathway enrichment analyses using DEGs in the indicated clusters. (L) Proportion of common myeloid progenitors (CMPs), megakaryocyte-erythroid progenitors (MEPs), steady-state GMPs (ssGMPs), and self-renewing GMPs (srGMPs) in control and Pcgf1?/? myeloid progenitors. *p<0.05; **p<0.01; ***p<0.001 by the Student’s t-test. Figure 4—source data 1.Raw data for Figure 4.