Mg 132 mg132

HeLa, hTERT-RPE1, U2OS, A549, DLD-1, and NIH/3T3 cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and streptomycin, and 2 mM L-glutamine at 37°C with 5% CO 2 . The HEC-6 cell line was cultured in DMEM supplemented with 15% FBS and 2 mM L-glutamine. Doxycycline-inducible cell lines were cultured in medium containing FBS certified tetracycline-free. spCas9 expression in inducible CRISPR/Cas9 cell lines was induced with 1 μg/ml doxycycline hyclate (Sigma) at 24 hr intervals for 2 days. All other doxycycline-inducible constructs were induced with 10 ng/ml doxycycline hyclate, unless indicated in figure legend. Other drugs used on human cells were Nocodazole (Sigma, 330 nM), S-trityl-L-cysteine (STLC; Sigma, 10 μM unless otherwise indicated), Paclitaxel (Taxol; Invitrogen, 1 μM), GSK923295 (CENP-E inhibitor; Selleck Chemicals, 100 nM), proTAME (APC/Ci; R&D Systems, 12 μM), AZ-3146 (Mps1i; Selleck Chemicals, 4 μM), cycloheximide (CHX; Sigma, 50 μg/ml), MG-132 (MG132; Enzo Life Sciences, 10 μM) unless concentration indicated otherwise in figure legend. Cells were enriched in mitosis with treatment with 330 nM Nocodazole for 16-17 hrs or if indicated, 10 μM STLC for 18 hrs. The antibodies used in this study are described in the relevant methods and in Supplementary Table 1.