Fig. 4 Brd4 binds and activates the BLIMP1 promoter
(A ) Effect of Brd4 inhibition on the expression of BLIMP1 in
human CD19+ B cells. Human CD19+ B cells were cultured with anti-IgM,
rCD40L, CpG ODN2006, rIL-10 and rIL-15 in the presence or absence of
PFI-1 or JQ1 for 7 days. BLIMP1 expression was measured by qRT-PCR. Data
are expressed as mean ± SEM from 5 independent experiments. (B )
Effect of Brd4 deficiency on expression of BLIMP1 in CD19+ B cells from
Brd4-CKO mice. Isolated splenic B cells were stimulated with LPS (10
μg/mL) and IL-4 (10 ng/mL) for 48 h. Data are expressed as mean ± SEM of
6 independent experiments. (C ) HEK-293T cells were
cotransfected with BLIMP1 reporter and Brd4 siRNA or control siRNA.
Luciferase activity was measured 48 hours post-transfection.
(D ) The potential Brd4 binding sites were evaluated by ChIP
assay using anti-Brd4 or anti-IgG antibodies in B220+B cells from spleen of wild type (FL/FL) mice. The presence of Brd4
binding site on BLIMP1 promotor was measured by qPCR.
*P <0.05, **P <0.01 versus control
(ctrl) or wild type (FL/FL) or siC,#P <0.05 versus co-stim.
Fig. 5 The effects of Brd4 inhibition on B cell differentiation
and Ig production in SLE. (A and B )
Purified CD19+ B cells from SLE patients (n=18) were cultured with
anti-IgM, rCD40L, CpG ODN2006, rIL-4 and rIL-2 as indicated in the
presence 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 evaluated after 7 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 (B ).
(C and D) The effects of Brd4 inhibitor
on differentiation of plasmablasts and plasma cells from SLE patients
(n=5). Purified 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 plasmablasts and plasma cells were examined
after 5 days in culture by flow cytometry. Data are mean ± SEM from five
independent experiments (D ). (E and F )
Effect of Brd4 inhibition on apoptosis (E ) and
proliferation (F ) of activated lupus CD19+ 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
PFI-1 for 7 days. For detection of apoptosis, cells were washed and
stained with annexin V and PI and analyzed by flow cytometry. For
measurement of proliferation, cells were analyzed for CFSE dilution.
(G ) IgG and IgM production detected by ELISA. The
results are the mean ± SEM from 8 independent experiments. *P <0.05 vernus no stim,#P <0.05 vernus co-stim without PFI-1
Fig 6 Effect of Brd4 inhibitor PFI-1 on hypergammaglobulinemia
and nephritis in MRL/lpr mice MRL/lpr mice (12 weeks of age)
were treated with vehicle (DMSO) or 2 mg/kg of PFI-1 for 6 weeks (n=8
mice per group). (A )
Effect of PFI-1 on s erum
levels of total IgG, IgG1, IgG2a and IgM. Serum samples obtained from
6-month-old MRL/lpr mice were used as the standard control.
(B and C ) Effect of PFI-1 on glomerulonephritis of
MRL/lpr mice. Representative kidney sections stained with PAS (B)
and frozen sections stained for IgG and C3 deposition (C). Original
magnification × 40. (D) Urinary protein levels in mice
at 12, 14, 16 and 18 weeks of age. (E) Serum creatinine (Cr)
and blood urea nitrogen (BUN) levels at the end of the experiment. Bars
in A, D and E show the mean ± SEM. *P<0.05 vs DMSO. (F and G )
Effect of Brd4 inhibitor PFI-1 on
the percentages of plasma cells
from spleen, lymph node and
peripheral blood mononuclear cells (PBMC) of MRL/lpr mice.
The cell surface markers of B cell
subsets were analyzed by flow cytometry. The numbers are the
percentages of cells within the quadrants. The bars represent the mean ±
SEM. *P <0.05 vs DMSO. (H ) Effect of PFI-1 on
the BLIMP1 expression of splenic B cell from MRL/lpr mice. BLIMP1
expression was determined by quantitative RT-PCR. Data represent the
mean ± SEM from 8 mice. *P <0.05, **P<0.01 vs DMSO. (I-K ) Effect of
Brd4 deficiency on nephritis and
percentages of plasma cells in Brd4-CKO mice with pristane-induced lupus
model. (I) Urinary protein levels in FL/FL mice (FL/FL, n=6),
pristane–injected CKO mice (CKO-SLE, n=6), and pristane–injected FL/FL
mice (FL/FL-SLE, n=6) at 0, 3 and 6 months after pristane injection.
(J ) Representative kidney sections from CKO and FL/FL mice
stained with hematoxylin and eosin (H&E) for analysis of glomerular
size and mesangial expansion. (K ) Effect of Brd4 deficiency on
frequency of plasma cell in mice with pristane-induced lupus model.
The frequency of plasma cells from
spleen (SP), lymph node (LN) and peripheral blood mononuclear cells
(PBMC) was evaluated using flow cytometric analysis. The numbers are the
percentages of cells within the quadrants. The bars represent the mean ±
SEM. *P < 0.05 versus FL/FL-SLE.