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: Membranes were blocked by incubation in WB blocking buffer (5% milk in TBS, 0.1% Tween) at RT for 1 h and probed with specific Abs diluted in WB blocking buffer or 5% BSA in TBS-T. Proteins were detected with antibodies against FIP200 (D10D11, AB_2797913), P62 (D1Q5S, AB_2799160), LC3B (D11, AB_2137707), cleaved caspase-3 (Asp175, AB_2341188), NLRP3 (D4D8T, AB_2722591), cleaved caspase-1 (Asp296) (E2G2I, AB_2923067) (Cell Signaling Technology), and TAX1BP1 (14424-1-AP) (AB_2198921; Proteintech) using the secondary HRP-conjugated anti-rabbit or anti-mouse antibodies (AB_2307391, AB_2338506; Jackson ImmunoResearch) or anti-β-actin−peroxidase antibody (AC-15) (AB_262011; MilliporeSigma).

Techniques: Staining, Clinical Proteomics, Expressing