Cancer cell assays MDA-MB-231 cells were grown in DMEM/F12 supplemented with 5% fetal
bovine serum and 5 μg/ml insulin. For the LysoTracker red assay, cells grown on coverslips were incubated with 100 nM LysoTracker red (Molecular Probes) for 25 min before addition of chemicals for 35 min. Cells were fixed with 3.7% paraformaldehyde in PBS, washed and DNA was stained with Hoechst 33342. For EGF internalization assays, cells grown on coverslips were incubated at 4°C for 1 h with 0.4 μg/ml FITC-EGF (Molecular Probes) in cell culture medium supplemented with buy Alisertib 2 mg/ml bovine serum albumin. Cells were then washed twice with cold medium before adding chemicals in cell culture medium at 37°C. After different times at 37°C, cells were SB273005 fixed with 3.7% paraformaldehyde in PBS, washed twice and mounted on slides for selleckchem microscopy. For EGFR immunostaining, cells grown on coverslips were fixed with 3.7% paraformaldehyde in PBS, permeabilized with 0.6% Triton X-100 in PBS, blocked with PBS containing 10% fetal bovine serum and 2% bovine serum albumin, incubated with 3 μg/ml monoclonal anti-EGFR antibody (Merck), washed and further incubated with CY3-conjugated goat anti-mouse IgG, F(ab’) fragment-specific antibody (Jackson Laboratory). Acknowledgements We thank Hilary Anderson for fruitful discussions, Martha Cyert for the genomic library, Raymond
Andersen and David Williams for motuporamines and Philip Hieter for the cyc3Δ yeast deletion strain. CN, GG and SH thank Ron Davis for providing the environment that allowed the development of the assays they contributed to this study. This work was supported by grants from the Canadian Institute of Health to GG (MOP-81340) and CN (MOP-84305), and by a Canadian Cancer Society grant through the National Cancer Institute of Canada to MR (017392). References
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