Figure S2.
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 NP29-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: SourceData FS2.

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 NP29-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: SourceData FS2.

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