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).