Fig 2: hnRNP F-deficient mice have normal B cell development.a, b Flow cytometric analysis of B cells in the BM. The frequencies (a) and numbers (b) of various B cell subsets in the BM of WT and Hnrnpf KO mice were analyzed and enumerated using Hardy’s gating strategy (n = 6 for WT group and n = 5 for Hnrnpf KO group). c, d Flow cytometric analysis of B cells in the spleen. The frequencies (c) and numbers (d) of different splenic B cell subsets in WT and Hnrnpf KO mice were examined and calculated (n = 6 for WT group and n = 5 for Hnrnpf KO group). Data are representative of five independent experiments (a and c). Each symbol represents an individual mouse (b and d). An unpaired two-tailed student’s t-test was used for the statistical analysis (b and d). Data are presented as mean values ± SD (b and d). Source data are provided as a Source Data file.

Fig 3: hnRNP F controls Cd40 AS.a Scatter plots of inclusion level difference between WT and Hnrnpf KO FOB cells. Each dot represents a significantly altered splicing event (|inclusion level difference | > 10%, FDR < 0.05). b A list of top ten genes with the highest aberrant AS events. c Relative expression of Hnrnpf and Cd40 mRNA in FOB cells based on the RNA-seq data (n = 2 per group). Data are from one single experiment. FDR was calculated by DESeq2 Bioconductor package based on the negative binomial distribution and and Benjamini–Hochberg method. d Sashimi plot of changes in AS of Cd40 exon 6-8 in WT and Hnrnpf KO FOB cells. The full-length version and different variants of Cd40 were shown. e The comparison of inclusion levels of indicated Cd40 AS events between WT and Hnrnpf KO FOB cells. FDR was calculated by rMATS. Likelihood-ratio test was used to calculate p value of the Inclusion Level between two groups. Benjamini Hochberg algorithm was used to correct p value to get FDR value (n = 2 per group). Data are from one single experiment. f Schematics of various isoforms of Cd40 mRNA generated by AS. The red lines above the exon indicate exon junctions, and the red line in CD40 V3 indicates CD40 intron 7. The red dotted line indicates exon sequences that are missing in CD40 V4 compared to CD40 V1. The arrows indicate the position of PCR primers for measuring Cd40 AS. g RT-PCR analysis of Cd40 variants using primers P1 and P2. The ratios of CD40 V2 to V1 and V6 to V1 were shown below the gel image. h RT-PCR analysis of Cd40 intron 7 retention using primers P3 and P4. The ratio of the variant with I7 retention to that without was shown below the gel image. Data are representative of three independent experiments (g) or two independent experiments (h). Data are presented as mean values ± SD (c and e). Source data are provided as a Source Data file.

Fig 4: hnRNP F is essential for GC B cell formation.a, b Flow cytometric analysis of the frequencies (a) and numbers (b) of total GC B cells in the spleen of WT and Hnrnpf KO mice (n = 6 per group) at day 10 post-immunization. c, d Flow cytometric analysis of the NP-specific IgG1+ B cells in the spleen of WT and Hnrnpf KO mice (n = 6 per group) at day 10 post-immunization. NP-specific IgG1+ B cells were determined by IgG1 and NIP+ BCR expression on the B220+Dump (CD138, IgM, CD3, IgD, Gr1)- cells (c). The frequencies and numbers of NP-specific IgG1+ B cells (d) were calculated accordingly. e–g Flow cytometric analysis of early GC B cell formation in the spleen of WT and Hnrnpf KO mice (n = 5 per group) at day 5 post-immunization. B220+GL-7+IgD-CD38- GC B cells were determined by flow cytometry (e), and their frequencies (f) and numbers (g) were summarized. h–j Flow cytometric analysis of iGB cells induced by anti-CD40, IL4, and IL21 for 4 days. B220+GL-7+Fas+CD38- iGC B cells were determined by flow cytometry (h), and their frequencies (i) and the Hnrnpf deletion efficiency (j) were summarized (n = 7 per group in (i) and n = 4 per group in j). k Analysis of proliferation of B cells stimulated with iGB condition for 3 days using cell trace dye. l qRT-PCR analysis of Myc expression in purified splenic B cells stimulated with iGB condition for 2 days. Data are representative of six independent experiments (a and c), three independent experiments (e, j, and k), four independent experiments (h), or one single experiment (l). Each symbol represents an individual mouse (b, d, f, g, i, j, and l). An unpaired two-tailed student’s t-test (b, d, f, g, i, and j) or two-way ANNOVA with Sidak’s multiple comparisons (l) was used for the statistical analysis. Data are presented as mean values ± SD (b, d, f, g, i, j, and l). Source data are provided as a Source Data file.

Fig 5: hnRNP F regulates GC B cell formation by promoting signalling-competent CD40 expression.a Flow cytometric analysis of the cell surface expression of CD40 on WT and Hnrnpf KO FOB cells (n = 3 per group). b Flow cytometric analysis of the cell surface expression of CD40 on WT and Hnrnpf KO GC (B220+CD38-FAS+) and non-GC (B220+CD38+FAS-) B cells. c Knockout efficiency of hnRNP F in Hnrnpf KO GCB cells with high and low CD40 expression using qRT-PCR (n = 4 for WT group and n = 3 for Hnrnpf KO group). d Flow cytometry analysis of the cell surface expression of CD40 on 293 T cells transfected with different CD40 variants. e, f Immunoblotting of phosphorylation of IKKα/IKKβ (e) and IκBα (f) in WT and Hnrnpf KO splenic B cells stimulated by 1 μg/ml anti-CD40 antibody. Data are from one single experiment (e and f). g The experimental flow of mixed BM chimeric mice study. h Flow cytometric analysis of the cell surface expression of CD40 on splenic B cells from different chimeric mice as indicated at day 10 after immunization with NP-CGG (n = 2 per group). i Flow cytometric analysis of GC B cells in the spleen of chimeric mice at day 10 after immunization (n = 4 for WT group and n = 6 for Hnrnpf KO group). Data are representative of two independent experiments (a, b, and d) or three independent experiments (h and i). Each symbol represents an individual mouse, and an unpaired two-tailed student’s t-test (a) or one-way ANOVA without adjustments (c, h, and i) was used for the statistical analysis. Data are presented as mean values ± SD (a, c, h, and i). Source data are provided as a Source Data file.