2.3. Homogenous Time Resolved Fluorescence (HTRF) Assay
The HTRF assay in 384-well plates (Greiner Bio-one, 784045) was performed according to the manufacturer’s instructions (Cisbio). Briefly, a total volume of 8 µL of BCL6-GST protein and 6His-SMRT peptide were added to each well, and then 2 µL of diluent buffer with the compounds to be tested was added. After 1 h, a total volume of 10 µL of anti-6His-XL665 (Cisbio) and anti-GST-Tb (Cisbio) was added to reach a 20 µL final volume. After overnight incubation, the plate was read on a microplate reader (BioTek Cytation5) at 665 nm and 620 nm.
2.4. Surface plasmon resonance (SPR) Assay
A Biacore T200 instrument (GE Healthcare) equipped with a CM5 sensor chip (GE Healthcare) was used to monitor binding interactions via SPR. The BCL6BTB protein was immobilized on CM5 using amine-coupling at a flow rate of 10 μL/min in 10 mM sodium acetate buffer (pH 4.0). Then, 0.1 mg/mL BCL6 BTB protein was injected for 420 s, and the surface was blocked by 1 M ethanolamine. The kinetics and affinity studies were performed in 25 mM HEPES, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20 and 5% DMSO at 25 °C. Various concentrations of compounds were injected into the flow system. Data analysis was performed using Biacore T200 Plus Evaluation Software (GE healthcare).
2.5. Cell culture and animals
Various cell lines were used including: 293T, SUDHL4, SUDHL6, OCI-Ly7, Farage, DOHH2, NCM460, PNT1A, LO2 and HAF. The cell lines SUDHL4, SUDHL6, Farage, NCM460, LO2, HAF and 293T were purchased from ATCC. OCI-Ly7 and DOHH2 were obtained from the DSMZ German collection of microorganisms and cell cultures and PNT1A was obtained from the ECACC. SUDHL4, SUDHL6, Farage, DOHH2, NCM460, PNT1A and LO2 were grown in RPMI-1640 medium (Gibco), and OCI-Ly7 was maintained in IMDM (Gibco). 293T cells were cultured in DMEM, and HAF cells were grown in DMEM with 2 mM glutamine. The medium was supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). All cells were incubated at 37 ℃ in a humidified atmosphere with 5% CO2.
All animals were obtained from the Animal Centre of East China Normal University. All procedures involving animals were approved by the Animal Investigation Committee of the Institute of Biomedical Sciences, East China Normal University.