Bi 605906

Validation of screen hits was conducted using MDA-MB-231 cells plated in
96 well plates. 90% confluent cells were stimulated with 100 nM PMA for
2 hours after which time supernatant was collected. A microsphere-based Luminex
Technology and ELISA kits (DY239, R&D Systems) were used to measure a
panel of shed growth factors, as previously described (49 (link)).
To measure changes in TGFα shedding with inhibitor treatments,
40,000 cells per well were plated in a 96 well plate, allowed to adhere
overnight and then incubated with serum-free medium for 3 hours. Cells were then
pre-treated for 1 hour with DMSO control or IRAK4 inhibitor (5uM AS2444697,
TOCRIS) or IRAK1/4 inhibitor (5 uM IRAK1/4 Inhibitor I, TOCRIS) or IKKb
inhibitor (2 uM BI605906, TOCRIS) or metalloprotease inhibitor (10uM Batimistat,
TOCRIS) after which medium was replaced with fresh media containing the
respective inhibitor and 100nM PMA (or vehicle). After 1 hour incubation,
TGFα shedding in the cell culture medium was quantified using the DuoSet
ELISA Development System (R&D systems). Cell viability was immediately
assessed using CellTiter-Glo Luminescent Cell Viability Assay (Promega).
TGFα shedding values (pg/mL) were normalized to cell viability
measurements.

Total CD4⁺ T cells were isolated from single-cell spleen suspensions using magnetic negative selection (eBioscience). Cells were stimulated at 37 °C, 5% CO₂ for 16 h in RPMI culture media supplemented with 10% fetal calf serum (Sigma), 1% penicillin and streptomycin (pen/strep; Gibco), and 50μM β-mercaptoethanol, non-essential amino acids (Gibco) and Glutamax (Gibco). Cells were plated at density of 1×10⁵ cells/well on 96-well plates in the presence or absence of 10ng/mL recombinant human IL-2 (Peprotech) with or without 300nM Tofacitinib (Sigma) or 10μM BI 605906 (Tocris). Where indicated, plates were coated overnight with 5 μg/mL anti-CD3 (clone 145.2C11; BioXcell Cat #BE0001-1) and 5 μg/mL anti-CD28 (clone 37.51 BioXcell Cat #BE0015-1) monoclonal antibodies in PBS before washing and plating of cells for stimulation. Cells were harvested and analysed by flow cytometry. To detect LPS-induced GARP expression on B cells, cells from erythrocyte-lysed blood were stimulated in RPMI complete media (RPMI, 10% FCS, 1% pen/strep, and 50μM β-mercaptoethanol) containing 0.5 mg/ml lipopolysaccharide (Sigma) at 37 °C, 5% CO₂ for 48 hr. At the end of the stimulation period, cells were collected and analysed using flow cytometry.

Purified B cells (0.5 million/sample) were treated with 5 nM of BAFF 3-mer or 60-mer proteins in a complete culture medium in 96 well plates for 20 hours, the cells were stained with 200 nM MitoTracker^™ Green FM (Fisher Scientific, M7514), 100 nM Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (Fisher Scientific, T669), and 2.5 μM Invitrogen^™ CellROX^™ Green Reagent (Fisher Scientific, C10444) in RPMI 1640 media for 20 minutes at 37 °C. LPS (1 μg/ml) was used as a positive control for B cell activation and 20 μM BAM15 was used as a negative control for dissipation of mitochondrial membrane potential detected by TMRE. The cells were pre-treated for 1 hour with a vehicle (complete culture medium with 0.1 % Dimethyl sulfoxide or various inhibitors of NF-κB signaling in 0.1% Dimethyl sulfoxide: BMS-345541 (5 μM, #16667, Cayman Chemicals, US), BI 605906 (5 uM, #5300, Tocris, US), and IMD 0354 (1 uM, #2611, Tocris) before treating with BAFF proteins. After the staining, the samples were run on the Flow cytometer machine Becton Dickinson LSRFortessa (equipped with laser lines 355nm, 405nm, 488nm, and 640nm). Dead cells were discriminated by using DAPI. Data analysis and quantification were performed using FlowJo v10.6.2.