Ppax2

All cell lines were tested for mycoplasma contamination and validated by STR DNA fingerprinting performed by the MDACC CCSG-funded Characterized Cell Line Core (NCI #CA016672). Human HEK 293 T, fibroblasts WI-38 and IM-R90 (ATCC), and human ovarian cancer cell lines CaoV3 and HeyA8 (gifts from Dr. Xiongbin Lu) were maintained in DMEM (Cellgro) supplemented with 10% fetal bovine serum (Sigma). Human lung cancer cell lines H1299, A549, H1355, Ludlu-1, and H520 (gifts from Dr. J. Heymach) were cultured in RPMI 1640 (Cellgro) supplemented with 10% fetal bovine serum. Retroviral or lentiviral transduction was performed as described below^{53 (link)}. Briefly, 293 T cells were co-transfected with pMD2.G, pPAX2 (Addgene), and pLKO shRNA or pCDH cDNA constructs. For infections, cells were incubated with viral supernatants in the presence of 8 µg/ml polybrene. After 48 h, the infected cells were selected with puromycin (2 µg/ml) for pLKO clones or blasticidin (10 µg/ml) for pCDH clones for 3–4 days before experiments.

MVT-1 cells were maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin mixture. To generate ZBP1 knockout (ZBP1 KO) MVT-1 cells, lentiviral sgRNA vector targeting ZBP1 was constructed by ligation of hybridized oligos into LentiCRISPR V2 (pXPR_001, GeCKO) vector. ZBP1 gRNA sequences are shown in Supplementary Table 1. 293T cells were transfected with sgRNA vector, pPAX2 (Addgene) and pCMV-VSV-g (Addgene) for 24 hr. MVT-1 cells were then infected with lentivirus-containing supernatants with polybrene (Millipore) for 24 h. After 3 days of selection with puromycin (2 µg ml⁻¹, Sigma-Aldrich), the ZBP1 KO cells were placed into 96 well plate to undergo clonal selection without puromycin. Single clones with complete knockout of ZBP1 was verified by DNA sequencing and western blot analysis.

Stable transfection of the ESRP1 (Sino Biological plasmid # HG13708-UT), CD44s, CD44v6, and NUMB1-4 (from VOR) expression plasmids was performed using FuGENE HD transfection reagent (Promega, E2311) according to the manufacturer’s protocol and selected with Geneticin (Gibco, 10131035). As for the KD constructs, the ESRP1-shRNA plasmid (Horizon, V3THS_335722) was packaged by pPAX2 (Addgene # 12260) and pMD2.G (Addgene # 12259) into HEK293T. The virus-containing supernatant was collected 24 hr after transfection, filtered, and employed to infect the HCT116 and SW480 cell line. Selection was applied with 750 ng/mL puromycin (Invivogen, San Diego, CA, USA) or 800 μg/mL of Geneticin selection for 1–2 weeks. The efficiency of overexpression and KD was assessed by qPCR and western blot 48–72 hr after transfection.

CRISPR screens were performed as previously described^{13 (link)}. Lentivirus was produced from the Brunello sgRNA library^{12 (link)} (Addgene 73178) in 293FT cells (Invitrogen) with helper plasmids pPAX2 (Addgene 12260) and pMD2.g (Addgene 12259) in a 4:3:1 ratio in Opti-Mem (Gibco) with Trans-IT 293 T (Mirus) following the manufacturer’s instructions. 293FT supernatants were harvested at 24, 48 and 72 h, pre-cleared by centrifugation at 1000 g for 5 min and concentrated by 40X using Lenti-X concentrator (Takara) following the manufacturer’s instructions. Concentrated Brunello lentiviral library was added to Cas9 MM clones to yield ~30% infection efficiency and maintain ~1 sgRNA per cell with an average of 500 copies per sgRNA in total. Infected MM cells were selected with puromycin 3 days after viral transduction and allowed to grow under selection for another 3 days. At this point, 50 × 10⁶ cells were harvested for the day 0 timepoint and 100 ng/ml of doxycycline and 0.5 μg/ml puromycin was added to at least 50 × 10⁶ cells to induce Cas9 expression, after which a minimum of 50 × 10⁶ cells were passed every other day for 21 days to maintain an average of 500X coverage/sgRNA in the Brunello library. 50 × 10⁶ cells were harvested for the day 21 timepoint. DNA was extracted from Day 0 and 21 cell pellets with QIAmp DNA Blood Midi and Maxi kits (Qiagen).