Str profiling

AU565 (ATCC CRL 2351) and MDAMB157 (ATCC HTB 24) cells were grown in DMEM supplemented with 10% FBS, HCC1143 (ATCC CRL 2321) cells were grown in RPMI supplemented with 10% FBS, and 21MT1 (generous gift from Kornelia Polyak) cells were grown in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml rhEGF, 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 µg/ml insulin. Cells were genetically engineered using plasmids available from Addgene. The coding fragment for clover-HDHB (Addgene plasmid 136461)^{23 (link)} was cloned in frame into a transposase expression plasmid modified to also express a nuclear localized mCherry and stable cell lines were created as previously described⁴⁴ . In brief, the clover-HDHB-NLS-mCherry expression plasmid was co-transfected 24–48 h with the pSB100X transposase plasmid (Addgene plasmid 34879) at a ratio of 4:1 using Lipofectamine 3000 (AU565, HCC1143, 21MT1) or LTX (MDAMB157) and selected for 3–7 days with 0.5–2 µg/ml puromycin. To ensure uniform fluorescence across the transfected population, HCC1143 and 21MT1 cells were sorted at OHSU’s Flow Cytometry Core and cells with a medium intensity clover-HDHB signal and a high intensity NLS-mCherry signal were selected for drug dose response experiments. In all cases, cells were validated by STR profiling (LabCorp) and tested negative for mycoplasma.

MDA-MB-231 (Cellosaurus CVCL_0062), PTXR231 (paclitaxel-resistant, Cellosaurus CVCL_4Z64) [52 (link)], RPE-1 (Cellosaurus CVCL_4388), HeLa (Cellosaurus CVCL_0030), and U2OS (CVCL_0042) cells were maintained in DMEM (Invitrogen) containing 10% FBS and 50 mg/mL penicillin/streptomycin (DMEM media). MCF10A (Cellosaurus CVCL_0598) cells were maintained in DMEM/F12 (Invitrogen, Waltham, MA, USA) containing 10% FBS, 50 mg/mL penicillin/streptomycin, 10 µg/mL insulin, 0.5X sodium pyruvate, 20 ng/mL EGF, and 0.5 µg/mL hydrocortisone (DMEM/F12 media). E8.1 HeLa cells were maintained in DMEM with 10% tetracycline-free FBS as previously described [53 (link),54 (link)]. To maintain cell-line integrity, cultures were not used for more than 20 passages. Identity of all cell lines was confirmed by STR profiling (LabCorp, Burlington, NC, USA). The relative expression level of MCAK in different cell lines was compiled from the Human Atlas project, the GSE10890 database and our own qPCR data (Table S1).

All cells were cultured at 37°; air; 95%; CO₂, 5%. H358 (NCI-H358), H23 (NCI-H23), H1792 (NCI-H1792) and 293T cells were obtained from American Type Tissue Culture (ATCC). Cells were regularly checked for mycoplasma contamination by polymerase chain reaction^{64 (link)} and found to be negative. H358 sgRB1#4 parental and resistant cell lines were verified by STR profiling (Labcorp, Burlington, NC, USA). LUAD cells were grown in RPMI−1640 medium (Gibco, 11875119) supplemented with 10% fetal bovine serum (FBS) (Gibco, 12483020) and 1% Pen/Strep (Gibco, 15140-122). 293T cells were grown in DMEM medium complete with 10% FBS and 1% Pen/Strep (Gibco, 15140-122). For cells and experiments with doxycycline-inducible constructs, cells were grown in RPMI-1640 medium (Gibco, 11875119) supplemented with 10% tetracycline-free FBS (Clontech, 631101) and Pen/Strep (Gibco, 15140-122). Doxycycline hyclate (Sigma-Aldrich, D9891) was added to cells at 200 ng/mL when indicated. Trametinib (Selleckchem, S2673), SCH772984 (Selleckchem, S7101), AMG 510 (Selleckchem, S8830), SB 747651 A (Tocris, 4630), MK-2206 (Selleckchem, S1078), NSC 23766 (Selleckchem, S8031), dabrafenib (Selleckchem, S8031), infigratinib (Selleckchem, S2183) and N-acetyl-L-cysteine (Sigma-Aldrich, A7250) were added to cells when indicated. Experiments were performed on cells between passages 4-20.