Figure 4.
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 IgDhi cells (DUMP−IgDhi) and plasma cells (DUMP−IgD−Sca-1+CD138+). Representative plots (F) and the corresponding quantifications (G) of IgDhi cells (top left), mROS of IgDhi 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: SourceData F4.

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 IgDhi cells (DUMP−IgDhi) and plasma cells (DUMP−IgD−Sca-1+CD138+). Representative plots (F) and the corresponding quantifications (G) of IgDhi cells (top left), mROS of IgDhi 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: SourceData F4.

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