Figure legends
Fig. 1 Effect of Brd4 inhibition on proliferation and apoptosis of activated human CD19+ B cells. (A ) Effect of Brd4 inhibitor on survival of activated B cells. Purified B cells were stimulated with combinations of anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2 as indicated in the presence of various concentration of Brd4 inhibitor PFI-1. At the time points indicated, the cells were stained with annexin V/PI. Percentages of viable cells (double negative for annexin V and PI) are shown as mean ± SEM of five independent experiments. (B and C ) Effect of Brd4 inhibitor on proliferation of activated B cells. Purified CD19+ B cells labeled with CFSE were cultured with combinations of anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2 in the presence of various concentration of PFI-1. After 7 days in culture, CD19+ B cells were analyzed for CFSE dilution. Panel B shown are representative of results. Data are mean ± SEM of five independent experiments (C ). (D and E ) Effect of Brd4 inhibitor on apoptosis of activated B cells. Purified B cells were cultured with no stimulus (nil), co-stimulation (anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2) in the presence of various concentrations of PFI-1. At the time points indicated, cells were washed and stained with annexin V and PI and analyzed by flow cytometry. The numbers in the lower right quadrants represent the percentage of apoptotic cells in culture (D ). Data are mean ± SEM of of five independent experiments (E ). *P <0.05 versus nil (no co-stimulation)
Fig. 2 Effect of Brd4 inhibition on B cell differentiation and Ig production. (A and B ) Purified human CD19+ B cells were cultured with anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2 as indicated in the presence of various concentration of Brd4 inhibitor PFI-1. The generation of naïve B cells (CD19+IgD+CD27-), memory B cells (CD19+IgD-CD27+), plasmablasts (CD19+IgD-CD27++) and plasma cells (CD19+CD38++CD138+) was assessed after 5 days in culture. The cells were stained for various surface markers and analyzed by flow cytometry. The numbers in the quadrants represent the percentage of cells in culture (A ). Data are mean ± SEM of five independent experiments (B ). (C and D ) Effect of PFI-1 on differentiation of plasmablast and plasma cells. Purified human CD19+ B cells were cultured with anti-IgM (5μg/ml), rCD40L (1μg/ml), CpG ODN2006 (5μM), rIL-10 (50ng/ml) and rIL-15 (10ng/ml). The percentage of plasmablast and plasma cells were examined after 5 days in culture by flow cytometry. Data are mean ± SEM of five independent experiments (D ). (E ) IgG and IgM production was detected by ELISA. The results are the mean ± SEM and are representative of five independent experiments. *P <0.05 versus nil (no co-stimulation), #P <0.05 versus co-stim without PFI-1
Fig. 3 The inhibitory effect of Brd4 deficiency on plasma cell differentiation in mice with deletion of Brd4 in CD19+ B cells.(A ) Schematic representation of the endogenous Brd4 locus, targeting vector, locus following homologous recombination, FLP-mediated deletion of the neomycin resistance cassette, and Cre-mediated deletion of start codon-containing exon 5. (B ) Genotyping the allele with loxP sites using the DNA templates isolated from mice tails. The loxP site was amplified with the primers F3 and R3; the 300-bp band indicates the allele with loxP sites, and the 211-bp band indicates the WT allele. (C ) The Brd4 CKO in CD19+ B cells was verified at the gene level with the splenic CD19+ B cells using RT-qPCR assay. (D-G ) Effect of Brd4 deficiency on differentiation of naïve B cells (CD19+IgD+CD27-, D and E ), memory B cells (CD19+IgD-CD27+, D and E ) and CD138+ plasma cells (F and G ). Isolated splenic CD19+ B cells were induced to differentiate using lipopolysaccharide (LPS, 10 μg/mL) and IL-4 (10 ng/mL) for 72 h. The percentage of naïve B cells, memory B cells and plasma cells were examined by flow cytometry. Data are mean ± SEM from 6 independent experiments (E and G ). The data are the mean ± SEM and are representative of 6 independent experiments.