Figure 1.

TRAF3 promoted the mitochondria-dependent intrinsic apoptotic pathway and was mainly localized at mitochondria in resting B cells. Splenic B cells were purified from gender-matched, young adult (8-12-week-old) naïve LMC or B-Traf3 ^-/- mice. Purified cells were analyzed directly (Day 0) or at day 2 after ex vivo culture in mouse B cell medium. (A) Representative FACS profiles of annexin V staining. Gated populations indicate the annexin V+ apoptotic cells. (B) Graphical results of the percentage of annexin V+ apoptotic cells. (C) Representative FACS profiles of cell cycle distribution analyzed by PI staining. Gated populations indicate apoptotic cells with DNA fragmentation (DNA content < 2n). (D) Graphical results of the percentage of apoptotic cells with DNA fragmentation. (E) Representative FACS profiles of mitochondrial membrane permeabilization measured by MitoProbe JC-1 staining. Gated populations in H show cells with healthy mitochondria and gated populations in P indicate cells with permeabilized mitochondria. (F) Graphical results of the percentage of cells with permeabilized mitochondria. (G) Cleavage of caspase 9 and caspase 3. Total cellular proteins were prepared at day 1 or day 2 after culture and immunoblotted for caspase 9 and caspase 3, followed by TRAF3 and actin. (H) Cytoplasmic TRAF3 proteins were mainly localized at mitochondria in resting B cells. ER, mitochondrial and S100 (cytosolic) proteins were biochemically fractionated and analyzed by immunoblotting. Proteins in each fraction were immunoblotted for TRAF3, calreticulin (an ER protein), COX IV (a mitochondrial protein) and actin. Immunoblots shown are representative of 3 experiments. Graphs shown in (B, D, F) are the mean ± SD (n=6/group, including 3 female and 3 male samples). ***p < 0.001 by t test.