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.