Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: B cell development in B- Fip200 −/− mice, related to Fig. 1. (A) Naïve B cells and CD4 + T cells were isolated from the spleens of WT or B- Fip200 −/− mice ( n = 3), and FIP200 expression was detected by western blot. (B and C) Flow cytometry analysis of BM (B) or spleen (C) from WT and Fip200 -KO-B mice. (B) BM samples were stained with antibodies against CD43, CD24, BP-1, IgM, IgD, and B220, and populations were identified following the Hardy classification system: B cells (B220 + ), early progenitors (B220 + CD43 + ), late progenitors (B220 + CD43 − ), fraction A (CD43 + CD24 − BP-1 − ), fraction B (CD43 + CD24 + BP-1 − ), fraction C (CD43 + CD24 + BP-1 + ), fraction D (CD43 − IgM − IgD − ), fraction E (CD43 − IgM + IgD − ), fraction F (CD43 − IgM + IgD + ). Left to right: **P = 0.0043, **P = 0.0043. (C) Spleen samples were stained with antibodies against CD21, CD23, CD24, and B220, and transitional or mature B cell populations were identified—B cells (B220 + ), T0-T1 cells (B220 + CD21 lo CD24 hi ), T2-MZB cells (B220 + CD21 hi CD24 hi ), MZB cells (B220 + CD21 hi CD23 lo ), follicular B cells (B220 + CD21 hi CD23 + ). Two replicates were performed with three to five animals in each group; one representative experiment is shown. Significant P values were determined by an unpaired t test. Top to bottom: *P = 0.0286, *P = 0.0286. Source data are available for this figure: .

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Isolation, Expressing, Western Blot, Flow Cytometry, Staining

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: B cell–specific FIP200-deficient mice showed an impaired humoral immune response. (A and B) Six WT and five B- Fip200 −/− mice were immunized with 30 μg NP 29 -KLH with Imject Alum and boosted at day 85 with 30 μg NP 29 -KLH in PBS. Mouse serum was collected throughout, and IC50 of anti-NP 29 (A)– or anti-NP 7 (B)–specific IgG antibodies was measured by ELISA. Serum collection days are marked on the x axes. (A) Left to right: *P = 0.0420, **P = 0.0098, **P = 0.0096, ***P = 0.0010 (unpaired t test). (B) Left to right; *P = 0.023, *P = 0.0102, **P = 0.0070, ***P = 0.0002 (unpaired t test). (C) Top: WT ( n = 5) and B- Fip200 −/− ( n = 3–7) mice were immunized with 50 μg NP 29 -KLH with Imject Alum and sacrificed at day 11. Bottom: Representative flow cytometry plots and frequency of Fas + CD38 − GC and CD138 + plasma B cells from two experiments; frequencies from one representative experiment quantified. *P = 0.0237, **P = 0.0028 (unpaired t test). (D) WT ( n = 9) and B- Fip200 −/− ( n = 6) mice were immunized with 30 μg NP 29 -KLH with Alhydrogel and sacrificed at day 42. Representative flow cytometry plots and frequency of B220 + CD38 + IgD lo NP + memory B cells from one experiment. Left to right: ***P = 0.0002, ***P = 0.0004 (unpaired t test). (E) At day 42, WT ( n = 4) and B cell–specific FIP200-deficient mice ( n = 4) were boosted with 30 μg NP 29 -KLH in PBS. Anti-NP 29 – and anti-NP 7 –specific IgG ASCs in both spleen and BM at day 49 by ELISPOT. Plots show values for individual mice (symbols) and mean ± SD (bars). Top: *P = 0.0255; bottom: *P = 0.0272, **P = 0.0031 (unpaired t test). Performed twice, one replicate shown.

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunospot

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: FIP200 is required for plasma but not GC maintenance. (A) 5 WT and 10 Aicda-Cre +/− FIP200-deficient mice were immunized with 50 μg NP 29 -KLH with Imject Alum and sacrificed at day 11. Two replicates were performed; representative flow cytometry plots and frequency of Fas + CD38 − GC and CD138 + plasma B cells from one experiment shown. (B) WT and Aicda-Cre +/− FIP200-deficient mice were immunized with 30 μg NP 29 -KLH with Imject Alum. Mouse serum was collected at regular intervals, and anti-NP 29 (upper)– or anti-NP 7 (lower)–specific IgG antibodies were detected by ELISA. Plots show values for individual mice (symbols) and mean ± SD (bars). Top: *P = 0.13; bottom: *P = 0.043. (C) WT ( n = 9) and Aicda - Cre +/− Fip200-deficient ( n = 8) mice were immunized with 30 μg NP 29 -KLH with Alhydrogel and sacrificed at day 42. Representative flow cytometry plots and frequency of B220 + CD38 + IgD lo IgG1 + NP + memory B cells from one experiment. Top to bottom: *P = 0.0111, **P = 0.0025 (unpaired t test). (D) WT ( n = 4) and Aicda-Cre +/− FIP200-deficient ( n = 4) mice were immunized with 30 μg NP 29 -KLH with Imject Alum and boosted with 30 μg NP 29 -KLH in PBS at day 42. Anti-NP 29 – and anti-NP 7 –specific IgG ASCs in BM at day 49 by ELISPOT. Plots show values for individual mice with triplicates. Significant (α = 0.05) P values were determined by an unpaired t test. Left to right: ***P = 0.0007, ***P = 0.0003.

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Clinical Proteomics, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: Fip200 −/− B cells showed decreased proliferation, plasma differentiation, and survival in vitro . B cells isolated from WT or B- Fip200 −/− mouse spleens were cultured with IL4 (10 ng/ml) + LPS (5 μg/ml). (A and B) Expression levels of p62 (A) and LC3 (B) were detected in WT and Fip200 −/− B cells upon stimulation at 0, 4, 16, 20, and 24 h. Representative data from one of at least two experimental replicates are shown. (C) Representative plots and the corresponding quantifications of plasma cells of WT and Fip200 −/− B cells after 3 days of culture. Data are combined from two independent experiments with at least three mice in each group, and samples are run in triplicate. Significant (α = 0.05) P values were determined by unpaired, two-tailed Student’s t test; ****P < 0.0001. (D) WT and Fip200 −/− B cells expressing Blimp-GFP were stimulated by IL4+LPS. Blimp-GFP + (day 2) or Blimp-GFP + CD138 + (day 3) populations were checked by FACS. N = 5 biological replicates, with representative data shown from one mouse. (E and F) Representative plots (E) and the corresponding quantifications (F) of the IRF4 hi PAX5 lo population of WT and Fip200 −/− B cells on days 1–3 of IL4 + LPS activation. Two independent experiments were performed. Representative data from one experiment with cells originating from four mice per group with two cultures generated per mouse are shown, except for KO, day 3, from which only one culture was obtained. Significant P values were determined by an unpaired t test. Left to right: ***P = 0.0002, ***P = 0.0002, **P = 0.0040. (G) Proliferation of WT and Fip200 −/− B cells upon stimulation by IL4+LPS was detected by FACS at days 1–3. Left to right: ***P = 0.0003, ***P = 0.0002. (H) Representative plots and the corresponding quantifications of IRF4 hi PAX5 lo population in proliferated populations (CTV lo ) of WT or Fip200 −/− B cells on day 2 of IL4 + LPS activation. Two independent experiments were performed. Representative data from one experiment with cells originating from four mice per group with two cultures generated per mouse are shown. Significant P values were determined by an unpaired t test. ***P = 0.0002. (I) WT and Fip200 −/− B cells were stimulated by IL4+LPS, and cell survival rate was detected by FACS on days 1–3. Left to right: **P = 0.0096, ***P = 0.0001, ****P < 0.0001 (unpaired t test). N = 3 biological replicates, with representative data shown from one mouse. (J) Cleaved caspase-3 was detected in WT and Fip200 −/− B cells after stimulation by IL4+LPS at days 2–3. N = 2 biological replicates, with representative data shown from one mouse. Source data are available for this figure: .

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Clinical Proteomics, In Vitro, Isolation, Cell Culture, Expressing, Two Tailed Test, Activation Assay, Generated

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: Fip200 −/− B cells show decreased proliferation and plasma differentiation, and increased dysfunctional mitochondria mass upon IL4+CD40L stimulation, related to Figs. 3 and 4. (A) Quantification of IgG1 + cells from WT and FIP200-deficient B cells cultured in the presence of IL4+LPS for 3 days. Data are combined from two independent experiments with four mice in each group, and samples are run in triplicate. Significant P values were determined by an unpaired t test. ****P < 0.0001. (B) Representative plots and the corresponding quantifications of plasma cells and IgG1 + of WT and FIP200-deficient B cells were cultured with IL4 (10 ng/ml) + CD40L (50 ng/ml) at day 3. Data are combined from two independent experiments with four mice in each group, and samples are run in triplicate. Significant P values were determined by an unpaired t test. ****P < 0.0001, *P = 0.0250. (C) Proliferation of WT ( n = 1–3) and Fip200 −/− ( n = 1–3) B cells upon stimulation of IL4+CD40L was detected by FACS at days 1–3. Three independent experiments were performed with representative data from one experiment with three mice per group shown. Left to right: ***P = 0.0002, ***P = 0.0008 (unpaired t test). (D and E) MD4 WT or Fip200 −/− B cells were stained with CTV, and OT-II T cells were stained with CFSE, then adoptively transferred to CD45.1 + recipient mice ( n = 5). Mice were immunized at day 1 with OVA-HEL by intravenous injection. Proliferation of OT-II T and MD4 cells B cells was detected by FACS at day 4. Left to right: *P = 0.0159, *P = 0.0159 (unpaired t test). (F) Cell survival rate of WT ( n = 1–3) and Fip200 −/− ( n = 1–3) B cells upon stimulation of IL4+CD40L was detected by FACS at days 1–3. Three independent experiments were performed with representative data from one experiment with three mice per group shown. (G) Cleaved caspase-3 was detected in WT and Fip200 −/− B cells upon stimulation by IL4+CD40L at days 1–3. Two biological replicates were performed with data from one mouse shown. (H) WT ( n = 5) and B- Fip200 -/- ( n = 4) mice were immunized with 50 μg NP 29 -KLH with Imject Alum and sacrificed at day 12. Quantifications of dead (Live/Dead Blue + Annexin V + ) and apoptosis (Live/Dead Blue − Annexin V + ) population in GC B cells (B220 + Fas + CD38 − ) by FACS. (I) Mitochondrial mass in day 3 IL4+CD40L-activated WT ( n = 2–5) and Fip200 −/− ( n = 3) B cells stained with MitoTracker Deep Red, ***P = 0.0001. Three independent experiments were performed with one shown. (J) OCR was measured by Seahorse XF analyzer ( n = 4) for activated B cells at day 1 (left) and day 2 (right). FCCP is a mitochondrial uncoupling agent. Oligo., oligomycin; R/A, rotenone/antimycin. Data are representative of at least two independent experiments with at least three mice in each group. Left to right: **P = 0.0079, **P = 0.0030, **P = 0.0016 (unpaired t test). (K and L) WT and Fip200 −/− B cells were stimulated by IL4+CD40L and stained with MitoSOX Red and gated on CD138 + population. Representative plots (K) and the corresponding quantifications (L) of total live cells (left) or plasma cells (right) of WT and Fip200 −/− B cells. Data are combination of two independent experiments with more than two mice in each group. Significant P values were determined by an unpaired t test. *P = 0.0125. Source data are available for this figure: .

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Clinical Proteomics, Cell Culture, Staining, Injection

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: Dysfunctional mitochondria accumulate in Fip200 −/− B cells. (A) Mitochondrial mass in naïve (left, WT, n = 3; and Fip200 −/− , n = 3) and day 3 IL4+LPS-activated (right, WT, n = 6; and Fip200 −/− , n = 3) B cells stained with MitoTracker Deep Red, ****P < 0.0001 (unpaired t test). Representative data of at least two experimental replicates shown. (B) OCR measured by Seahorse XF analyzer for activated B cells (four samples per group) at day 0 (left), day 1 (middle), and day 2 (right). FCCP is a mitochondrial uncoupling agent. Oligo., oligomycin; R/A, rotenone/antimycin. From left to right: day 0, *P = 0.0325, **P = 0.0014, ***P = 0.0007,**P = 0.0022, **P = 0.0083, **P = 0.0026, **P = 0.0057; day 1, **P = 0.0027, **P = 0.0041, **P = 0.0051, **P = 0.0058, ***P = 0.0002, ***P = 0.0003, ***P = 0.0004; day 2, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001 (unpaired t test). (C) Splenocytes from WT ( n = 6) and B- Fip200 −/− ( n = 5) mice were stained with MitoSOX Red and naïve splenic B cells (left) and B220 − cells (right) detected by FACS, **P = 0.0022 (unpaired t test). (D and E) WT and Fip200 −/− B cells were stimulated by IL4+LPS and stained with MitoSOX Red and gated on CD138 + population. Representative plots (D) and the corresponding quantifications (E) of total live cells (left) or plasma cells (right) of WT and Fip200 −/− B cells. Data are representative of at least two independent experiments run with two mice per group. Representative data from one experiment are shown. Left to right: ****P < 0.0001, ****P < 0.0001 (unpaired t test). (F and G) BM cells from WT ( n = 5) and B- Fip200 −/− ( n = 6) mice were stained with MitoSOX and gated on IgD hi cells (DUMP − IgD hi ) and plasma cells (DUMP − IgD − Sca-1 + CD138 + ). Representative plots (F) and the corresponding quantifications (G) of IgD hi cells (top left), mROS of IgD hi cells (top right), plasma cells (bottom left), or mROS of plasma (bottom right) of WT and Fip200 −/− B cells. Two independent experiments were performed; representative data from one experiment are shown. Top left to right: *P = 0.0173, *P = 0.0173; bottom left to right: *P = 0.0173, **P = 0.0043 (unpaired t test). (H) Expression level of NLRP3 in WT and Fip200 −/− B cells and cleaved caspase-1 in the supernatant upon IL4+LPS stimulation at day 2. This experiment was performed twice, with results from one independent run shown; each lane represents one mouse. Significant (α = 0.05) P values were determined by an unpaired t test. *P = 0.0182. Source data are available for this figure: .

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Staining, Clinical Proteomics, Expressing

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: FIP200 regulates mitophagy in B cells upon activation. (A) Schematic breeding strategy to generate MitoQC-B- Fip200 −/− mice and their littermate controls. (B) Dynamic of the MitoQC-GFP expression level in WT and MitoQC-B- Fip200 −/− B cells on day 3 of IL4+LPS stimulation. Data are representative of at least three independent experiments ( n = 2–3 mice per treatment). One representative experiment is shown. (C) Representative cells from one WT mouse to demonstrate BDS scoring approach in gate R4 (GFP + mCherry + ). (D) Representative plots of BDS scores of total live cells and CD138 + plasma cells measured by ImageStream; data were analyzed by the Kolmogorov–Smirnov test, ****P < 0.0001. Cells were cultured independently from two WT mice, with summary data from one mouse-derived culture shown. (E) Representative plots of BDS scores of WT and MitoQC-B- Fip200 −/− B cells on day 3 of IL4+LPS stimulation; data were analyzed by the Kolmogorov–Smirnov test, ****P < 0.0001. The experiment was performed at least three times ( n = 1–3); representative data from cultures derived from one WT and one knockout mouse are shown here.

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Activation Assay, Expressing, Clinical Proteomics, Cell Culture, Derivative Assay, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: FIP200 mediates mitophagy of TAX1BP1 binding mitochondria in B cells upon CD40L stimulation, related to Fig. 5. (A) Dynamic of the MitoQC-GFP expression level in WT and MitoQC-B- Fip200 −/− B cells upon IL4+CD40L stimulation for 3 days. Data are representative of at least two independent experiments with three mice in each group. (B) Gating strategy of Live/single/focused/GFP + mCherry + cells in ImageStream. (C) Representative plots of BDS score of MitoQC-B cells treated with FCCP or control (DMSO) for 30 min. Data are representative of at least two independent experiments; data from naïve B cells from one mouse per treatment are shown. (D) Representative plots of BDS score of WT and MitoQC-B- Fip200 −/− B cells upon IL4+CD40L stimulation for 3 days; data were analyzed by the Kolmogorov–Smirnov test, ****P < 0.0001. The experiment was performed independently three times, with one representative experiment shown. n = 2–3. (E) Expression of TAX1BP1 was detected in WT and Fip200 −/− B cells upon IL4+CD40L stimulation for 3 days. Two biological replicates were performed for WT and Fip200 −/− ; results from one shown. (F) Representative immunofluorescence Airyscan images with 90x magnification (Plan-Apochromat 50×/1.2 W objective, 1.8× magnification changer) of WT ( n = 2; one shown) and MitoQC-B- Fip200 −/− ( n = 2; one shown) B cells activated by IL4+CD40L at day 2 and stained with anti-TAX1BP1 antibody (blue), MitoQC-GFP (green), and MitoQC-mCherry (red). Scale bar, 3 μm. Yellow arrows point to TAX1BP1 aggregation in B cells. Source data are available for this figure: .

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Binding Assay, Expressing, Control, Immunofluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: Sample preparation for 10x single-cell RNA-Seq and cluster definition of B cells from WT and B- Fip200 −/− mice, related to Fig. 6. (A) Gating strategy of FACSorted plasma, GC, and memory B cell populations in IgD + cell–depleted splenocytes from WT and B- Fip200 −/− mice. Naïve B cells (B220 + IgD hi ) were sorted from intact splenocytes. (B) Statistical analysis of GC, plasma, and memory B cell populations in IgD + cell–depleted splenocytes from WT and B- Fip200 −/− mice, as in . Significant P values were determined by an unpaired t test. Left to right: *P = 0.0316, *P = 0.0405, *P = 0.0268. (C) Heat map of top five expressed genes in 13 clusters in .

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Sample Prep, RNA Sequencing, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: FIP200 promotes plasma differentiation by balancing ROS and heme metabolism. (A) Naïve, GC, memory, and plasma B cells FACSorted from WT and B- Fip200 −/− mice immunized with 50 μg NP 29 -KLH with Imject Alum at day 11. UMAP showing 13 B cell clusters and phenotypic identities in WT (left) and Fip200 −/− (right) groups. Representative of one experiment, n = 5 pooled B- Fip200 −/− mice and n = 4 pooled WT mice. (B) Venn diagram shows common genes upregulated in Fip200 −/− cells in clusters 2, 6, and 7 (major plasma populations), and genes upregulated in all three clusters compared with their WT control. (C) Subset and reclustering of B cells from plasma clusters 2, 6, 7, and 12 in A, colored by cluster. (D) Scatterplots showing average signature score, calculated in VISION, for curated KEGG pathways on a cluster-by-cluster basis in Fip200 −/− versus WT plasma cells for ROS (left) and heme metabolism (right). FDR, false discovery rate; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Clinical Proteomics, Control

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: Single-cell RNA-Seq of different B cell populations from WT and B- Fip200 −/− mice, related to Fig. 6. (A) UMAP of 13 B cell clusters and phenotypic identities in merged WT and Fip200 −/− groups in . (B and C) Feature plot of the B cells in A showing relative expression of signature genes labeled on top (B) and antibody-derived tag (ADT) antibody expression (C). Color scale represents centered natural log transformation across cells. (D) Distribution of immunoglobulin subclass mapped in the plasma population UMAP representation. The color scale represents centered natural log transformation across cells. (E) Plot of nucleotide mutation counts in the heavy chain variable region of different plasma clusters. Feature plot of cells for their CITE-Seq/ADT antibody expression. (F) NP-specific IgG1, IgG2b, IgG2c titers and IgA antibody level were detected by ELISA at day 56 in WT ( n = 7) or B- Fip200 −/− ( n = 8) mice after 30 μg NP 29 -KLH with Imject Alum immunization. Plots show values for individual mice (symbols) and means (bars). Significant P values were determined by an unpaired t test. Top: ***P = 0.0003; bottom: **P = 0.0093, ***P = 0.0003.

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: RNA Sequencing, Expressing, Labeling, Derivative Assay, Transformation Assay, Clinical Proteomics, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: FIP200 regulates plasma B cell differentiation via mitochondrial and heme homeostasis

doi: 10.1084/jem.20250535

Figure Lengend Snippet: Hemin rescued plasma differentiation in Fip200 −/− B cells. (A) Quantification of the hemin level in WT ( n = 2–3) and Fip200 −/− ( n = 3–4) B cells at the naïve stage and day 3 after activation. P values were determined by an unpaired t test. **P = 0.0072. Three independent experiments were performed, with representative data shown for one. (B) Quantification of plasma cell fractions from WT and Fip200 −/− B cells cultured in the presence of IL4+CD40L (left), and IL4+LPS (right) with or without hemin (60 μM) for 3 days. Three independent experiments were performed; data from one experiment are shown ( n = 1–3 per treatment/genotype). Left: ***P = 0.0004, right: ***P = 0.0004 (C) Representative plots of WT and Fip200 −/− B cells expressing Blimp-GFP were stimulated by IL4+CD40L (left) and IL4+LPS (right) without hemin (upper) or with hemin (60 μM, bottom) at day 3 by FACS. Three biological replicates were performed for each treatment; data from one experiment are shown. (D) mROS level in WT and Fip200 −/− B cells in the presence of IL4+LPS with or without hemin (60 μM). Two independent experiments were performed with three mice in each group; data from one experiment are shown.

Article Snippet: B cells isolated from MitoQC-Fip200 f/f Mb1-Cre −/− or MitoQC-Fip200 f/f Mb1-Cre +/− mice using a Pan B cell isolation kit (Miltenyi) were activated by IL4+CD40L for 2 days.

Techniques: Clinical Proteomics, Activation Assay, Cell Culture, Expressing