Journal: Frontiers in Immunology

Article Title: Mitochondrial calcium shapes B cell signaling and mitochondrial function

doi: 10.3389/fimmu.2025.1710128

Figure Lengend Snippet: The loss of MCU alters cell signaling and oxygen consumption. (A–G) Mcu fl/fl x mb1 CreERT2 (MCU -/- ) and control (Ctrl) mice were injected with tamoxifen for three consecutive days to induce B cell-specific Mcu deletion. (A) Representative immunoblot analysis of MCU protein levels in MCU -/- and Ctrl mouse B cells stimulated with anti-IgM for 2 days. Representative of 3 independent experiments. (B) Mitochondrial Ca 2+ levels in MCU -/- and Ctrl mouse B cells stimulated with anti-mouse IgM (10µg/ml) for 1 (left) and 2 days (right) were determined by Rhod-2 AM staining. Pooled data from 6 and 5 independent experiments, respectively. (d1: n=9 for MCU -/- and 10 for Ctrl, d2: n=8 for MCU -/- and Ctrl mice). (C) Measurement of mouse B cell proliferation by eFluor 670 dilution. MCU -/- and Ctrl mouse B cells were stimulated with anti- IgM and tracked for 3 days. Representative of 3 independent experiments. (n=6 MCU -/- and 5 Ctrl mice). (D) Cell survival analysis of mouse B cells shown in (C) . Forward scatter (FSC) and side scatter (SSC) properties were used to determine the percentage of living cells. Pooled data from 3 experiments. (n=6 MCU -/- and 5 Ctrl mice). (E) Cytosolic Ca 2+ was assessed in MCU -/- and Ctrl B cells using indo-1 AM. Shown are basal and peak Ca 2+ levels after anti-IgM stimulation as well as the difference between peak and basal levels (delta). Pooled data from 5 independent experiments. (n=7 for MCU -/- and for Ctrl). (F) Representative immunoblot analysis of NFAT2 protein levels in MCU -/- mouse B cells compared to Ctrl B cells. Cells were stimulated with anti-mouse IgM (10µg/ml) for 48 h. Blots were probed for NFAT2 and actin. Representative of 3 independent experiments. (G) Quantification of (F) was performed by normalization of NFAT2 levels to actin levels. Then, fold change in NFAT2 levels of MCU -/- mouse B cells relative to Ctrl B cells was calculated. Pooled data from 3 independent experiments. (n=5 Ctrl and 7 MCU -/- mice). (H–N) Mcu fl/fl and Ctrl B cells were treated with TAT-CRE and stimulated with anti-IgM. (H) Representative immunoblot analysis of MCU protein levels in MCU -/- and Ctrl mouse B cells stimulated with anti-IgM for 48h. Blots were probed with MCU and actin antibodies. Representative of 3 independent experiments. (I) Mitochondrial Ca 2+ levels in MCU -/- and Ctrl mouse B cells stimulated as in (H) was determined with Rhod-2 AM staining. Pooled data from 5 independent experiments. (n=6 MCU -/- and 7 Ctrl mice). (J) MCU -/- and Ctrl B cells were stimulated with anti-IgM. Proliferation was assessed by eFluor 670 dilution. Representative of 3 independent experiments. (K) Representative immunoblot analysis of NFAT2 protein expression in MCU -/- and Ctrl mouse B cells shown in (H) . Blots were probed for NFAT2 and actin. Representative of 3 independent experiments. (L) Quantification of (K) was performed by normalization of NFAT2 levels to actin levels. Then, fold change of NFAT2 levels of MCU-deficient mouse B cells relative to Ctrl B cells was calculated. Pooled data from 3 independent repeats. (n=4 for MCU -/- and Ctrl mice). (M) Representative oxygen consumption measurement of MCU -/- and Ctrl mouse B cells as in (H) . Oxygen consumption rates (OCR) were measured by Seahorse flux technology after sequential injection of oligomycin (1µM), FCCP (1µM) and rotenone + antimycin A (1µM). Representative of 3 independent experiments. (N) Summary graphs showing calculated basal and maximal respiration in anti-IgM stimulated MCU-deficient and Ctrl mouse B cells shown in (M) . Pooled data from 3 independent experiments. (n=3 for MCU -/- and Ctrl). Data are presented as mean. Paired (B, I, N) and unpaired (G, L) Student’s t tests were used to compare groups. *p < 0.05; ns, not significant.

Article Snippet: Mcu fl/fl mice (B6;129S- Mcu tm1.1Jmol /J, The Jackson Laboratory) were crossed to mb1-CreER T2 mice (B6.C- Cd79a tm3(cre/ERT2)Reth /EhobJ, The Jackson Laboratory).

Techniques: Control, Injection, Western Blot, Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T-bet + B cells Dominate the Peritoneal Cavity B Cell Response During Murine Intracellular Bacterial Infection

doi: 10.4049/jimmunol.2101209

Figure Lengend Snippet: T-bet-cre ERT2 x Rosa26 EYFP mice were fed tamoxifen chow from days 6-9 post-infection; on day 9 post-infection peritoneal cavity cells were analyzed by flow cytometry and transferred at a 1:1 ratio to the peritoneal cavity of mice infected on the same day. Two days later, the recipient mice were sacrificed and their B cells were analyzed by flow cytometry. Cells (3 × 10 6 ) were stained from each site and analyzed to repletion on the cytometer. Correction for organ cellularity and sampling is shown in Supplementary Table 2 . (A) Flow cytometric analysis of T-bet expression among donor peritoneal cavity B cells on day 9 post-infection gated for singlet live dump-neg (CD4/CD8/Ter119) cells is shown. A representative of 2 biological replicates is shown. (B) Flow cytometric analysis of T-bet expression among recipient peritoneal cavity B cells on day 11 post-infection gated for singlet live dump-neg (CD4/CD8/Ter119) cells is shown. A representative of 2 biological replicates is shown. (C) Flow cytometric analysis of T-bet expression among recipient splenic B cells on day 11 post-infection gated for singlet live dump-neg (CD4/CD8/Ter119) cells is shown. A representative of 2 biological replicates is shown.

Article Snippet: C57BL/6J, Tbx21 fl (B6.129-Tbx21 tm2Srnr /J), Mb1 cre (B6.C(Cg)-Cd79a tm1(cre)Reth /EhobJ), and Rosa26 eYFP (B6.Cg- Gt(ROSA)26Sor tm3(CAG-EYFP)Hze /J) mice were obtained from The Jackson Laboratory (Bar Harbor, ME).

Techniques: Infection, Flow Cytometry, Staining, Cytometry, Sampling, Expressing