Treatment of MRL/lpr mice with the Brd4 inhibitor PFI-1
Female MRL/lpr mice were purchased from Shanghai SLAC Laboratory Animal
Center (Chinese Academy of Sciences). 12 week-old female MRL/lpr mice
were randomly divided into 2 groups (8 mice per group). The two groups
of mice were intraperitoneally injected daily with PFI-1 (10 mg/kg/d) or
vehicle.
Mouse urine was collected every week for 24 h in metabolic cages
starting from week 12. Urinary protein, blood urea nitrogen (BUN) and
creatinine (Cr) concentrations were determined by assay kits (Jiancheng
Biotechnology, Nanjing, China) according to the manufacturer’s
instructions.
The serum levels of anti-dsDNA antibodies, ANA, complement C3,
complement C4, IgG, IgG1, IgG2a and IgM were assayed using ELISA kits
(USCN LIFE Science Inc, Wuhan, China) according to the manufacturer’s
instructions. The percentages of plasma cells
(CD19+CD138+), naïve B cells
(CD19+IgM+IgD+CD27-)
and memory B cells
(CD19+IgM+IgD-CD27+)
were detected by flow cytometry.
Kidney tissues were collected and immersed in 10% neutral buffered
formalin immediately. Paraffin sections were stained with haematoxylin
and eosin (HE). Histopathology of glomerular and renal vascular lesions
was graded on a scale of 0-3 for severity in a blinded manner.
For the detection of immune complex deposition, kidneys were embedded in
OCT compound and snap-frozen at -80°C. Frozen kidney sections (5 μm)
were fixed in acetone, blocked with 10% goat serum for 30 minutes, and
stained with fluorescein isothiocyanate (FITC)-conjugated goat
anti-mouse IgG or mouse complement C3 (Cedarlane, Burlington, Ontario,
Canada).