Fig 2: 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 3: 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 4: The bPHA uncovers class-specific kinetics of Syk recruitment to BCR.(A) Schematic diagrams showing that upon stimulation, Syk can be recruited to CD79a. The proximity between CD79a and Syk can be measured by bPHA using oligo-coupled anti-CD79a and anti-Syk antibodies. (B) Anti-CD79a+:anti-Syk− bPHA signals were measured by flow cytometry for resting and anti-IgD- or anti-IgM-stimulated splenic B cells. Cells without the corresponding target binding probes served as controls for both the resting and stimulated cells. Data represent three independent experiments. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; IgD, immunoglobulin D; IgM, immunoglobulin M; Syk, spleen tyrosine kinase.

Fig 5: Dietary fibers and SCFAs alter AID and Blimp1 expression, bacteria-bound/binding antibody responses, and gut bacteria composition.C57BL/6 mice fed a fiber or no-fiber diet were on SCFAs water (SCFAs) or plain water (nil) and administered OVA together with CT (same as in Fig. 1). a, b AID and Blimp1 expression in spleen B cells of these mice, AID expression in spleen B cells of Aicda-/- mice, and Blimp1 expression in naive B cells of C57BL/6 mice, as analyzed by intracellular staining followed by fluorescence microscopy (a) and flow cytometry (b). c Quantification of AID+ and Blimp1+ cells in a and b. n = 3 per group. d Bacteria-bound IgM, IgD, IgG1, and IgA in the feces of fiber diet-fed C57BL/6 mice on SCFAs water or plain water, collected 1 week after the last OVA and CT administration, as well as bacteria-bound IgA in the feces of unimmunized Aicda–/– mice fed fiber diet and plain water, as analyzed by flow cytometry. e Bacteria-bound IgA in feces of fiber and no-fiber diet-fed mice on SCFAs water or plain water as analyzed by flow cytometry. f Concentration of bacteria-binding IgM and IgA in the serum as measured by ELISA; each symbol represents an individual mouse (n = 5 per group, pooled from two experiments). The bars represent the mean ± SD. g Relative abundance of gut bacteria classes (> 1% in any sample group; fiber, no-fiber, and no-fiber + SCFAs) in the mice (n = 4 per group, pooled from two experiments) as determined by high-throughput 16s rRNA gene miSeq sequencing. h Principal component analysis of gut bacterial composition in these three groups of mice on different diets. Data in a, b, d, and e are representative of three independent experiments. *p < 0.05, ***p < 0.001 values, ns: not significant (unpaired t test). Scale bar = 100 µm. The source data are provided in Source Data file.