Selumetinib or trametnib (Sellekchem) were added to tumor cells in
96-well plates. Viability was measured using the CellTiter Glo luminescent
viability kit (Promega) after 72 hr using a Wallace plate reader (Molecular
Probe). To determine pERK after MEKis treatment, Kras mutant lung tumor cell
lines and splenocytes collected from HKP1 tumor bearing mice were treated
with selumetinib or trametinib for 2 hr at 37C. Selumetinib and trametinib
stock solutions were prepared in DMSO and diluted in media. Cells were
stimulated with 0.1 ug/ml PMA for 2 min at 37C, then immediately fixed with
4% paraformaldehyde and permeabilized using ice cold 95% methanol. pERK was
stained using anti-pERK (#9106, Cell signaling) and goat anti-mouse
Ig(H+L)–FITC (SouthernBiotech) by flow cytometry. Washout experiments
were done using CD5+ splenocytes from HKP1 tumor bearing mice. CD5+ cells
were collected using CD5 microbeads (Miltenyi MACS cell separation system),
then seeded in the presence of selumetinib or trametinib into flat bottom
96-well plates pre-coated with anti-CD3 (145–2C11, 1ug/ml) and
anti-CD28 (37N, 1ug/ml). After 24hrs, media (RIPA + 7.5% FBS + 0.1%
b-mercaptoethanol) was replaced with MEKis (continuous group) or DMSO
diluent (wash out group) and the media was changed every day with freshly
prepared MEKis. At 72hrs and 96 hr, cells were collected and analyzed by
flow cytometry.
96-well plates. Viability was measured using the CellTiter Glo luminescent
viability kit (Promega) after 72 hr using a Wallace plate reader (Molecular
Probe). To determine pERK after MEKis treatment, Kras mutant lung tumor cell
lines and splenocytes collected from HKP1 tumor bearing mice were treated
with selumetinib or trametinib for 2 hr at 37C. Selumetinib and trametinib
stock solutions were prepared in DMSO and diluted in media. Cells were
stimulated with 0.1 ug/ml PMA for 2 min at 37C, then immediately fixed with
4% paraformaldehyde and permeabilized using ice cold 95% methanol. pERK was
stained using anti-pERK (#9106, Cell signaling) and goat anti-mouse
Ig(H+L)–FITC (SouthernBiotech) by flow cytometry. Washout experiments
were done using CD5+ splenocytes from HKP1 tumor bearing mice. CD5+ cells
were collected using CD5 microbeads (Miltenyi MACS cell separation system),
then seeded in the presence of selumetinib or trametinib into flat bottom
96-well plates pre-coated with anti-CD3 (145–2C11, 1ug/ml) and
anti-CD28 (37N, 1ug/ml). After 24hrs, media (RIPA + 7.5% FBS + 0.1%
b-mercaptoethanol) was replaced with MEKis (continuous group) or DMSO
diluent (wash out group) and the media was changed every day with freshly
prepared MEKis. At 72hrs and 96 hr, cells were collected and analyzed by
flow cytometry.