Fig 2: Mof exhibits dynamic expression along erythropoiesis, and its reduction has a pronounced impact on erythroid lineage commitment.(A) Heatmap representing gene expression patterns of KATs and HDACs along the erythroid trajectory [data from (18)]. (B) Top: Schematic representation of fate-bias analysis, showing the cell fate-bias score (x axis) versus transcript expression (y axis). The fate-bias score indicates the probability of a given cell to belong to target cell types A, B, or C. A score of 1.0 indicates that the given cell has 100% probability of belonging to a target cell type. The example cell expressing Gene X has a 75 to 100% chance of belonging to cell type A but only a 0 to 20% chance of belonging to cell types B or C. In the analysis represented in the bottom panel, we selected erythroid, myeloid, and lymphoid cells as our target cell types. Bottom: Dot plot from 4763 cKit+ cells, showing the cell fate-bias score determined by FateID plotted against RaceID-normalized transcript expression in erythroid [Gata1+Klf1+Gypa+Hba-a1+; cluster 3; erythroid versus Mof expression r = +2 × 10-2; Pearson correlation, P = 4 × 10-2), myeloid (Mpo+Elane+; cluster 1; myeloid versus Mof expression r = -3.6 × 10-2; Pearson correlation, P = 6.3 × 10-3), and lymphoid (Ebf1+Rag1+; cluster 7; lymphoid versus Mof expression r = +6 × 10-3; Pearson correlation, P = 0.32) lineages; original data from (12)]. For t-distributed stochastic neighbor embedding (t-SNE), maps, and cluster annotation see fig. S1 (A and B). (C) Box plot showing the total number of RBC, hemoglobin (HB) quantity, and hematocrit (HCT) percentage (n = 4 per genotype) and scatter plot showing the HB concentration measured using an enzyme-linked immunosorbent assay (ELISA)–based assay (wild type, n = 7; Mof+/-, n = 13; P < 0.001). (D) Left: Box plot showing the total area of the colonies obtained from the single-cell colony-forming unit (sc-CFU) assay on fluorescence-activated cell sorting (FACS)–sorted HSCs (LSK+Flt3-CD34-CD48-CD150+). Right: Box plot showing the total cell number per colony obtained in the sc-CFU assay. (E) Pie charts representing the lineage potency from the sc-CFU assay. (F) Stacked bar plot showing the fraction of lineage output from each clone. Final populations were defined on the basis of cell surface markers: myeloid (Mye; cKit-CD11b+, number of cells: 208,961 wild type and 905,455 Mof+/-), erythroid (Ery; cKit-Ter119+, number of cells: 205,963 wild-type and 27,564 Mof+/-), lymphoid [Lym; cKit-IgD+, number of cells: 65,093 wild-type and 63,815 Mof+/-], and progenitor cells (Prog; cKit+, number of cells: 98,988 wild-type and 124,690 Mof+/-). Significant enrichment by Fisher’s exact test was set as P > 0.05. sc-CFU data from 72 wild-type and 147 Mof+/- colonies from three independent animals. (G) Bar plots showing the CFU capacity output of 100 FACS-sorted HSCs (wild-type, n = 6; Mof+/-, n = 3). (H) Same as (G), but using HSCs sorted from Cag-Cre:ERT2Tg/+(control) or Moffl/flCag-Cre:ERT2Tg/+transgene (Mof-iKO) animals. After sorting, cells were cultured with 4-hydroxytamoxifen (4-OHT) to induce Mof depletion in vitro. Bar plots show CFU capacity output of HSCs (control, n = 3; Mof-iKO, n = 3). (I) Serial CFU assay scheme and serial colony formation capacity from (G) or (H). (J) CFU assay scheme and CFU capacity output of FACS-sorted MEPs (Lin-cKithighSca-1-IL-7R-CD34-FcRgII/III-) from wild-type or Mof+/- (wild-type, n = 6; Mof+/-, n = 3) animals. (K) same as (J), but from control or Mof-iKO–sorted MEPs, wherein Mof depletion was induced in vitro after sorting and plating. Error bars represent means ± SEM, and biological replicates are represented as the overlaid dots. Experimental significance was determined by one-way analysis of variance (ANOVA), P < 0.05. Related to figs. S1 to S4.

Fig 3: The bPHA works in a mixed cell population.(A) Schematic diagram showing the route map for generating GFP-µm-expressing IgM or IgD KO cells from WT Ramos B cells by CRSPR/Cas9 method. (B) Flow cytometry results showing that the subpopulations of the mixed cells can be identified by gating for the expression of GFP and anti-IgD staining. (C and D) TD05+:TD05- (C) or Enh+:Enh- (D) bPHA results were measured by flow cytometry and analyzed by the gating strategy shown in (B). Data represent three independent experiments. bPHA, branched proximity hybridization assay; Enh, Enhancer; GFP, Green fluorescent protein; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out; PE, phycoerythrin; WT, wild-type.

Fig 4: The bPHA confirms the rearrangement of BCR upon stimulation.(A) TD05+:TD05-, TD05+:Enh-, and Enh+:Enh- bPHA signals were measured by flow cytometry for resting and stimulated IgM-KO GFP-µm-expressing Ramos cells. The stimulated cells without the corresponding target binding probes served as control. Data represent four independent experiments. (B) Schematic diagrams showing that on the surface of IgM-KO GFP-µm-expressing Ramos cells surface, upon stimulation, IgD-BCR and GFP-µm mix together, producing positive TD05+:Enh- bPHA signal. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; Enh, Enhancer; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out.

Fig 5: Dietary fibers and SCFAs dampen CSR, plasma cell differentiation and class-switched antibody responses.After weaning, C57BL/6 mice were fed a fiber diet (regular chow) or no-fiber diet. Two weeks later, these mice were started on SCFAs water (SCFAs) or plain water (Nil). All mice were then administered OVA together with CT via intragastric gavage once a week for 4 weeks starting at 8 weeks of age, and killed a week after the last OVA and CT administration. a Total and OVA-binding IgM, IgG1, IgA, and IgE concentrations in serum of fiber or no-fiber-fed mice on SCFAs water or plain water, as measured by ELISA. Each symbol represents an individual mouse (n = 5 per group, pooled from two experiments). The bars represent the mean ± SD. b OVA-binding IgM, IgG1, IgA, and IgE concentrations in the feces of fiber-fed mice on SCFAs water or plain water, as measured by ELISA. Each symbol represents an individual mouse (n = 5 per group, pooled from two experiments). c IgM, IgA, and IgD-positive cells in the lamina propria, PPs, and MLNs of fiber or no-fiber-fed mice on SCFAs water or plain water as visualized by fluorescence microscopy. d ELISPOT analysis of IgM, IgG1, and IgA AFCs in MLNs, PPs, spleen, and bone marrow of fiber-fed mice on SCFAs water or plain water. e Proportion of surface CD19+IgG1+ and CD19+IgA+ B cells, germinal center B cells (CD19+GL-7+), and plasmablasts/plasma cells (CD138+) in MLNs, PPs, and the spleen of fiber-fed mice given SCFAs water or plain water, as analyzed by flow cytometry. Data in c–e are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (unpaired t test). Scale bar = 100 µm. The source data are provided in Source Data file.