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.