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.
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.