Glutamax

CMT-93 cells, a murine rectal-carcinoma cell line obtained from ATCC, were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, 100 ug/ml of streptomycin and 2 mM GlutaMAX (all reagents obtained from Gibco). Cells were passaged (1:10) after reaching 70~80% confluence. sgRNA design and cloning were performed based on a protocol by Ran et al. (34 (link)), with a CRISPR design tool (http://crispr.mit.edu). The sequences of oligos were: Forward primer: 5′-CACCgCCATCGGATGTCCTCTGCGC-3′, Reverser primer: 5′AAACGCGCAGAGGACATCCGATGGC-3′. ATF3 targeting sequences were cloned into BbsI-digested pSpCas9(BB)-2A-GFP plasmid (Addgene, px458), which was a gift from Dr. Liuh-Yow Chen at Academia Sinica, Taiwan. The px458-based constructs were introduced into CMT-93 cells by PolyJet^TMin vitro DNA Transfection Reagent using advanced protocol (SignaGen). Single GFP⁺ cells were sorted into 96-well plate to obtain single cell clones by FACSAria cell sorter (BD Biosciences) at IBMS core facility, Academia Sinica, Taiwan. The ATF3-deficient cells were confirmed by Western blot analysis.

The human epithelial colorectal adenocarcinoma cell line, Caco-2, was kindly donated by Jan Trige Rasmussen (Aarhus University, Denmark). Cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Denmark) supplemented with 10% fetal calf serum (FCS, Bio-Whittaker, Denmark), HEPES (10 mM, Life Technologies), GlutaMax (2 mM, Life Technologies), and penicillin-streptomycin (1% v/v, Sigma-Aldrich, Denmark). RAW264.7 macrophage cells (ATCC, Germany) were cultivated in DMEM supplemented with 10% FCS, 2 mM GlutaMax, and 1% v/v penicillin-streptomycin. Cells were maintained at 37°C and 5% CO₂. Caco-2 cells were passaged using 0.05% Trypsin-EDTA (Life Technologies) and RAW264.7 cells were passaged by scraping off cells when reaching 80–90% confluence.

Cell culture was performed as reported previously^{23 (link)} and reiterated here. COS-7 cells were obtained from ATCC (Manassas, VA, USA, catalog number CRL-1651) and maintained in standard DMEM-HG medium supplemented with 10% (vol/vol) heat-inactivated FBS, 1 mM sodium pyruvate and 2 mM Glutamax in a 5% CO₂ incubator at 37 ^°C. For imaging, the cells were plated in Bioptechs Delta-T dishes (Bioptechs, Butler, PA, USA, product number 04200417B) and grown to ~ 70% confluency. Transient transfections were done with various plasmids according to manufacturer’s instructions using 1–2 μg DNA and 3ul of X-tremeGene HP DNA transfection reagent (Roche, Manheim, Germany, product number 06366236001). The transfected cells were incubated at 37 °C and 5% CO₂ incubator for 24–48 h before imaging. The cell culture reagents DMEM-HG medium (catalog number 11960), fetal bovine serum (FBS, catalog number 10082), sodium pyruvate (catalog number 11360) and Glutamax (catalog number 35050) were obtained from Invitrogen (Life Technologies, Grand Island, NY, USA). Calyculin A was obtained from Sigma (catalog number C 5552).

SEM cells, a KMT2A-AFF1 B cell ALL line (Greil et al. 1994 (link)), were purchased from DSMZ (https://www.dsmz.de). SEM cells were cultured in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal bovine serum (FBS) and 1× GlutaMAX, with cell density maintained between 5 × 10⁵/mL and 2 × 10⁶/mL. RS4;11 and THP-1 cells were purchased from ATCC (https://www.atcc.org). RS4;11 and THP-1 cells were cultured in RPMI-1640 with 10% FBS and 1× GlutaMAX, with cell density maintained between 5 × 10⁵/mL and 1.5 × 10⁶/mL. Cells were confirmed to be free of mycoplasma.