PC3 (CRL-1435), AML12 (CRL-2254), and HepG2 (HB-8065) cells were purchased from the American Type Culture Collection (ATCC). PC3 cells transduced with lentivirus expressing shRNA against FKBP12 or FKBP51 or a nonspecific control were thawed from Kennedy laboratory stocks30 (link) and reselected in puromycin prior to experimentation. PC3 and HepG2 cells were cultured in DMEM (Fisher Scientific, MT10013CV) containing 10% FBS (Sigma, 12303C) and 1% penicillin–streptomycin (Fisher Scientific, MT30002CI). AML12 cells were cultured in DMEM/F-12 media (Fisher Scientific, MT10090CV) with 10% FBS and 1% penicillin–streptomycin.
Mt30002ci
Manufactured by Thermo Fisher Scientific
The MT30002CI is a laboratory equipment product by Thermo Fisher Scientific. It is a core laboratory instrument designed for specific functions. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti vinculin, by Cell Signaling Technology (2 mentions)
Hepg2, by Thermo Fisher Scientific (1 mentions)
Hepg2 hb 8065, by American Type Culture Collection (1 mentions)
Mt10013cv, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Raw 264.7 atcc tib 71, by American Type Culture Collection (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Mt10013cv, by Thermo Fisher Scientific (1 mentions)
Plko 1 blast scramble, by Addgene (1 mentions)
F2442 500ml, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Crl 1573, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Complete dmem media, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Mycoalert assay, by Lonza (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
8 protocols using mt30002ci
1
FKBP12 and FKBP51 Knockdown in PC3 Cells
2
Culturing Mouse Macrophages and Human Fibroblasts
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The mouse macrophage cell line RAW 264.7 (ATCC TIB-71, obtained from American Type Culture Collection, Livingstone, MT, USA) was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Grand Island, NY, USA), supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, NY, USA), not exceeding 80% confluency at 37°C in a humidified incubator with 5% CO2. Primary human dermal fibroblasts isolated from adult skin (HDFa, Invitrogen C-013-5C) were cultured in Medium 106 (Invitrogen M-106-500) with Low Serum Growth Supplement (LSGS, Invitrogen S-003-10) and antibiotic penicillin/streptomycin solution 100 IU/100 μg/ml (Fisher MT-30-002-CI).
3
FOXR1 Fusion Dependency Validation
4
Genetic Manipulation of Breast Cancer Cell Lines
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MCF-7 cells were maintained in RPMI media (11875093, Invitrogen) and MDA-MB-231 and 293T in DMEM media (MT10013CV, Fisher Scientific). Media contained 10% FBS (F2442-500ML, Sigma-Aldrich) and Pen-Strep (MT30002CI, Fisher Scientific) and cells were grown under standard tissue culture conditions. Cells were split using Trypsin (MT25053CI, Fisher Scientific) according to manufacturer's instructions.
MDA-MB-231 cells overexpressing WT HOTAIR, A783U mutant HOTAIR, or Anti-Luciferase were generated as previously described using retroviral transduction (Meredith et al., 2016) (link). Stable knockdown of METTL3, METTL14, WTAP, and YTHDC1 and overexpression of YTHDC1 was performed by lentivirus infection of MCF-7 or MDA-MB-231 cells overexpressing HOTAIR or A783U mutant HOTAIR via Fugene HD R.8 with pLKO.1-blasticidin shRNA constructs or a pLX304 overexpression construct as noted in Table S3. Cells were selected with 5 µg/mL blasticidin (Life Technologies). The nontargeting shRNA pLKO.1-blast-SCRAMBLE was obtained from Addgene (Catalog #26701). Two shRNAs for each target were obtained and stable lentiviral transductions with the targeted shRNAs and the scramble control were performed. Cell lines with the most efficient knockdown as determined by western blot were selected for downstream experiments.
MDA-MB-231 cells overexpressing WT HOTAIR, A783U mutant HOTAIR, or Anti-Luciferase were generated as previously described using retroviral transduction (Meredith et al., 2016) (link). Stable knockdown of METTL3, METTL14, WTAP, and YTHDC1 and overexpression of YTHDC1 was performed by lentivirus infection of MCF-7 or MDA-MB-231 cells overexpressing HOTAIR or A783U mutant HOTAIR via Fugene HD R.8 with pLKO.1-blasticidin shRNA constructs or a pLX304 overexpression construct as noted in Table S3. Cells were selected with 5 µg/mL blasticidin (Life Technologies). The nontargeting shRNA pLKO.1-blast-SCRAMBLE was obtained from Addgene (Catalog #26701). Two shRNAs for each target were obtained and stable lentiviral transductions with the targeted shRNAs and the scramble control were performed. Cell lines with the most efficient knockdown as determined by western blot were selected for downstream experiments.
5
Immunoprecipitation and Immunoblotting in HEK293 Cells
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Expression of the constructs, coimmunoprecipitation, and protein immunoblots were carried out as described (Senften et al., 2006 (link); Zhao et al., 2016 (link); Liu et al., 2018 (link)). In brief, HEK293 cells (CRL-1573, RRID:CVCL_0045; ATCC) were cultured in DMEM supplemented with 10% heat-inactivated FBS (MT35011CV; Thermo Fisher Scientific) and 1% penicillin/streptomycin (MT30002CI; Thermo Fisher Scientific). Lipofectamine 2000 (11668019; Life Technologies) was used to transfect plasmids into HEK293 cells. 2 d after transfection, cells were harvested and lysed using radioimmunoprecipitation assay buffer containing 50 mM Hepes, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitors. Then supernatant was collected after centrifugation at 13,000 rpm for 15 min at 4°C. Immunoprecipitations were then carried out using anti-HA agarose beads (E6779, RRID:AB_10109562; Sigma-Aldrich). After washing, immunoprecipitated proteins were eluted by boiling in SDS sample buffer.
6
Culturing Human Hepatocellular Carcinoma HepG2 Cells
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Human hepatocyte carcinoma cells (HepG2) were grown in complete DMEM media (31053036, Thermo Fisher Scientific) with 10% Avantor Seradigm premium grade fetal bovine serum (97068-085, VWR), 1 mM sodium pyruvate (11360070, Thermo Fisher Scientific), 100 IU penicillin and 100 μg/ml streptomycin (MT30002CI, Thermo Fisher Scientific), and 2 mM L-glutamine (25-030-081, Gibco). The cells were subcultured every 2–3 days at 70% confluence. The HepG2 cells were gifted to us from Dr. Patricia Oteiza’s laboratory. All experiments were performed on cells between passages 10 and 20. Sterile culturing and assay plates were used for the following experiments. Cells were regularly tested for mycoplasma every 6 months using the MycoAlert assay (LT07-701, Lonza).
7
Cell Culture Preparation and Delipidation
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The neuroblastoma cell lines Neuro-2a, SK-N-SH and IMR-32 were purchased from American Type Culture Collection. The hepatocyte carcinoma cell line HepG2 and lung carcinoma cell line A549 were purchased from the European Collection of Authenticated Cell Cultures (via Sigma-Aldrich). All cell lines were maintained as recommended by distributors, subcultured every 4–5 d up to 10 passages, and culture medium was changed every 2 days. All media components were purchased from Thermo Fisher Scientific Inc., including minimum essential medium eagle with glutamine (MEM, MT100010CV), Dulbecco’s modified eagle medium with glucose and sodium pyruvate without glutamine (DMEM, MT15013CV), L-glutamine (25-030-081), penicillin-streptomycin solution (P/S, MT30002CI) and N-2 Supplement (17-502-048). Premium grade fetal bovine serum (FBS) was obtained from VWR/Seradigm (97068-085). Delipidated FBS (dFBS) was prepared by stirring of silica (S5130, Sigma-Aldrich) into of FBS for 24 h at 4°C followed by centrifugation for 1 h at at 4°C in a Thermo Scientific Sorvall RC-6 Plus superspeed centrifuge. Supernatant was collected and validated for delipidation of cholesterol using UPLC-MS/MS derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD).
8
Validating FOXR1 Fusion Dependencies
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143B (obtained from the Broad Institute; used within 6 months of collection; authenticated by L. Guenther using STR profiling; no mycoplasma testing conducted) and CALU6 (obtained from ATCC; used within 6 months of collection; authenticated by ATCC using STR profiling; mycoplasma testing conducted by ATCC, mycoplasma not detected) cells were grown in Eagle's minimum essential medium (EMEM) supplemented with 10% FBS and penicillin–streptomycin (Thermo Fisher Scientific, MT30002CI). To validate FOXR1 fusion dependencies, we used CRISPR-Cas9 sgRNAs targeting FOXR1 to knockout PAFAH1B2-FOXR1 in 143B cells and RPS25-FOXR1in CALU6 cells. The sgRNA sequences are as follows: sgFOXR1–1 5′ GAGACCTCCAGCTTTCCAGG 3′; sgFOXR1–2 5′ GGAAGATGCCAGCTGCTCAG 3′; sgFOXR1–3 5′ TGAGACCTCCAGCTTTCCAG 3′. We infected cells with either nontargeting (NT) or FOXR1-targeting sgRNA, selected cells with puromycin (1 μg/mL), and assessed growth using CellTiter-Glo according to manufacturers' instructions. Immunoblot was performed to confirm knockout of the fusion-oncoproteins using anti-FOXR1 (21942–1-AP; Thermo Fisher Scientific). Anti-vinculin (13901; Cell Signaling Technology) was used as a loading control. For crystal violet staining, cells were fixed and stained using crystal violet solution (1% crystal violet, 20% methanol) for 20 minutes, washed, and imaged.
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