Hek279T, MDA-MB-231, LM2, BT549, Cal51, Cal120 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 9% fetal bovine serum (Sigma), 2mM glutamine, 0.1mg/ml penicillin and 0.1ml/ml streptomycin (Gibco). HCC1806, HCC38, Hs578T cells were maintained in RPMI supplemented with glutamine.
Hek293T cells were used for lentivirus production as described previously [19 (link)]. shRNAs targeting EGFR and ROCK were obtained from the TRCs1.0 library and were as follows: shEGFR-1: TRCN0000121068, shEGFR-2: TRCN0000010329, shEGFR-3: TRCN0000121206, shEGFR-4: TRCN0000121203, shROCK1-1: TRCN0000002163, shROCK1-2: TRCN0000121316, shROCK1-3: TRCN0000121095, shROCK1-4: TRCN0000002161 (TRC Library, Sigma).
For long-term cell growth assays, cells were seeded on 6-well or 12-well plates (Corning). Drugs were added on the following day and media was refreshed every third day with new compound dilutions. At the end time point, the cells were stained with crystal violet. ROCK inhibitors used were GSK269962A (Axon) and Fasudil (Selleck). EGFR inhibitors used were Gefitinib (MedChem) and Afatinib (Selleck).
For DNA content and cell cycle analysis, sub-confluent cells were incubated with 10uM Bromdeoxyuridine (BrdU) for 1.5 hours, trypsinized, fixed in 70% ice-cold ethanol, and stained with anti-BrdU and Propidium Iodide (PI).
Hek293T cells were used for lentivirus production as described previously [19 (link)]. shRNAs targeting EGFR and ROCK were obtained from the TRCs1.0 library and were as follows: shEGFR-1: TRCN0000121068, shEGFR-2: TRCN0000010329, shEGFR-3: TRCN0000121206, shEGFR-4: TRCN0000121203, shROCK1-1: TRCN0000002163, shROCK1-2: TRCN0000121316, shROCK1-3: TRCN0000121095, shROCK1-4: TRCN0000002161 (TRC Library, Sigma).
For long-term cell growth assays, cells were seeded on 6-well or 12-well plates (Corning). Drugs were added on the following day and media was refreshed every third day with new compound dilutions. At the end time point, the cells were stained with crystal violet. ROCK inhibitors used were GSK269962A (Axon) and Fasudil (Selleck). EGFR inhibitors used were Gefitinib (MedChem) and Afatinib (Selleck).
For DNA content and cell cycle analysis, sub-confluent cells were incubated with 10uM Bromdeoxyuridine (BrdU) for 1.5 hours, trypsinized, fixed in 70% ice-cold ethanol, and stained with anti-BrdU and Propidium Iodide (PI).
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