HeLa T-REx cells (Invitrogen) were grown and passaged in DMEM supplemented with 10% FBS. Xenopus S3 cells were cultured in 66% L-15 media (Sigma-Aldrich) supplemented with 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 1 nM sodium pyruvate at 18°C. HeLa cells were plated at 25% confluency onto poly-l -lysine–coated 18-mm coverslips in a 12-well dish (Corning) overnight. siRNA transfection for EB1 and Mad2 knockdown was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol, and survivin knockdown was performed using Oligofectamine (Invitrogen) as previously described (Niedzialkowska et al., 2012 (link)). HeLa cells were treated with EB1 siRNAs (Custom siRNA, 5′-AAGUGAAAUUCCAAGCUAAGCUU-3′; Custom 3′UTR siRNAs, 5′-GAATGCTGGAGAGATGTTATG-3′ and 5′-GCACTAATCTCTTTGGAGA-3′; Thermo Fisher Scientific) at 10 nM and a SMARTpool of siRNA oligonucleotides against Mad2 (L-003271-00-0005; Thermo Fisher Scientific) with a final concentration of 20 nM, either separately or in combination for 48 h. pDLAP-EB1 (wild type or siRNA resistant) was transiently transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol, 24 h after siRNA transfection, for rescue experiments. Cells grown in a 12-well dish were transfected with 250 ng (500 ng for a 6-well dish) plasmid and fixed for immunofluorescence after 24 h.
T rex hela cells
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The T-REx-HeLa cells are a cell line derived from human cervical cancer cells that have been engineered to express the tetracycline repressor (TetR) protein. This cell line allows for the tetracycline-regulated expression of genes of interest.
Automatically generated - may contain errors
Lab products found in correlation
Transdux reagent, by System Biosciences (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
L 15 media, by Merck Group (1 mentions)
12 well dish, by Corning (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Culture flask, by Corning (1 mentions)
Heracell 150i co2 incubator, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Psuper retro neo gfp, by Oligoengine (1 mentions)
Doxycycline hyclate, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Bodipy red, by Thermo Fisher Scientific (1 mentions)
14 protocols using t rex hela cells
1
Investigating EB1 and Mad2 Knockdown in HeLa Cells
2
Stable Transfection of HeLa Cells with hTDO
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HeLa T-REx cells were purchased from Invitrogen (Karlsruhe, Germany) and stably transfected with pcDNA4-hTDO vector (Invitrogen, Karlsruhe, Germany) containing human liver TDO cDNA to produce HeLa-hTDO cells as described by us [4 (link)]. The expression of recombinant hTDO in HeLa-hTDO cells was induced by stimulation with tetracycline.
The cells were cultured in Iscove's modified Dulbecco's medium (IMDM) (Gibco, Grand Island, USA), supplied with 5% heat-inactivated fetal calf serum (FCS) in culture flasks (Costar, Cambridge, USA) and split weekly in 1 : 10 ratios by using trypsin/EDTA (Gibco, Grand Island, USA). Mycoplasma contamination was regularly excluded via PCR. Hypoxia growth experiments were carried out using alternatively a HERAcell 150 I CO2 incubator (Thermo Fisher Scientific, Langenselbold, Germany) or the Anoxomat™ system (Mart Microbiology B.V., Drachten, Netherlands) with 1–10% O2 and 10% CO2. The IMDM was buffered with sodium bicarbonate and therefore had an optimal buffering capacity at the 10% CO2 environment to maintain the physiological pH. Consequently the pH values of the cell culture medium with or without cells under normoxia and hypoxia were in the range of 7.15–7.48.
The cells were cultured in Iscove's modified Dulbecco's medium (IMDM) (Gibco, Grand Island, USA), supplied with 5% heat-inactivated fetal calf serum (FCS) in culture flasks (Costar, Cambridge, USA) and split weekly in 1 : 10 ratios by using trypsin/EDTA (Gibco, Grand Island, USA). Mycoplasma contamination was regularly excluded via PCR. Hypoxia growth experiments were carried out using alternatively a HERAcell 150 I CO2 incubator (Thermo Fisher Scientific, Langenselbold, Germany) or the Anoxomat™ system (Mart Microbiology B.V., Drachten, Netherlands) with 1–10% O2 and 10% CO2. The IMDM was buffered with sodium bicarbonate and therefore had an optimal buffering capacity at the 10% CO2 environment to maintain the physiological pH. Consequently the pH values of the cell culture medium with or without cells under normoxia and hypoxia were in the range of 7.15–7.48.
3
Inducible Expression of Yeast tRNA3-Phe in HeLa Cells
4
Generation of GFP-Nup98 and GFP-Nup98-HOXA9 cell lines
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pcDNA4-GFP-Nup98 and pcDNA4-GFP-Nup98-HOXA9 were generated by BamHI/EcoRI digestion of pEGFP-Nup98 and pEGFP-Nup98-HOXA9, respectively. The GFP-Nup98 and GFP-Nup98-HOXA9 fragments included a stop codon at the end of the sequence and were inserted into BamHI/EcoRI cut pcDNA4-TO-myc-His B (Invitrogen). HeLa T-Rex cells (Invitrogen) were grown in minimal essential medium (MEM) containing GlutaMAX™ (Invitrogen) supplemented with 10% FBS (Biochrom), 100 U/ml penicillin and 100 U/ml streptomycin (Invitrogen) and transfected with pcDNA4-GFP-Nup98 or pcDNA4-GFP-Nup98-HOXA9 using Turbofect. Cells were incubated for 48 h and diluted 1:10 into fresh MEM. After another 24 h, the medium was exchanged by fresh medium containing 5 μg/ml blasticidin (Invitrogen) and 200 μg/ml zeocin (Invivogen, Toulouse, France). After 2 to 3 weeks, single colonies were selected and tested for expression of GFP-Nup98 or GFP-Nup98-HOXA9 by addition of 1 μg/ml tetracycline (Sigma–Aldrich) for 24 h and subsequent analysis by Western blotting. Selected clones were maintained stable in MEM containing 5 μg/ml blasticidin and 200 μg/ml zeocin.
5
HeLa T-REx Cell Transfection and Lipid Starvation
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HeLa T-REx cells were obtained from Thermo Fisher Scientific (catalog: R71407). They were confirmed to be free of mycoplasma once per month. Cells were cultured in DMEM supplemented with 10% FBS (Gibco) and maintained in 5% CO2 at 37°C. Cells were transfected with Lipofectamine 2000 Transfection Reagent (Invitrogen) using 40–50 ng of plasmid DNA (unless otherwise indicated) mixed with 1 µl of Lipofectamine 2000 and cultured for 16–20 h prior to analysis. For PISD siRNA experiments, a TriFECTa Kit DsiRNA Duplex kit (accession number: NM_014338.3; Hs.Ri.PISD.13.1: 142688286; Hs.Ri.PISD.13.2: 142688283; Hs.Ri.PISD.13.3: 142688280) was used according to the manufacturer's instructions (Integrated DNA Technologies).
For the lipid starvation experiment shown inFig. 2 , transfected cells were treated with 100 µM oleic acid and 0.5 µM BODIPY Red (Invitrogen) for 24 h. Cells were washed three times with PBS and then incubated in media supplemented with 5% delipidated fetal bovine serum (Gemini BioProducts) for 0, 24 and 48 h.
For the lipid starvation experiment shown in
6
Stable Expression of Borealin Variants in HeLa T-REx Cells
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HeLa T-REx cells (ThermoFisher Scientific) stably expressing LAP-Borealinwt, LAP-BorealinLLPS, and LAP-BorealinMTBM (19 (link)) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco) in the presence of 5% CO2 in a humidified incubator at 37 °C.
7
Localization Analysis of Dyrk2 Variants
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T-REx-HeLa cells (#R71407, Invitrogen) expressing GFP-tagged Dyrk2 variants were fixed on coverslips for 10 min with 600 μl 4 % Paraformaldehyde. After washing with PBS fixed cells were permeabilized with 0.2 % Triton-X 100 for 5 min, washed with PBS and then incubated with 600 μl 2% BSA for 30 min. Nuclear staining was performed with Hoechst (1:10,000, 10 mg/ml stock solution). Coverslips were mounted on the microscope slide with ProLong Gold antifade reagent (Invitrogen).
For the analysis of localization of Dyrk2 WT, KO, and SX mutant, more than 20 cells per condition were imaged on a wide-field Olympus MM microscope. Images were analyzed in Image J Fiji v1, cytoplasmic and nuclear signals were obtained by manual and automatic segmentation, respectively, and their ratio calculated.
For the analysis of localization of Dyrk2 WT, KO, and SX mutant, more than 20 cells per condition were imaged on a wide-field Olympus MM microscope. Images were analyzed in Image J Fiji v1, cytoplasmic and nuclear signals were obtained by manual and automatic segmentation, respectively, and their ratio calculated.
8
Stable Cell Line Generation of Ndc80 Tail Mutants
9
MTT Assay for Cell Viability
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T-REx-HeLa cells (#R71407, Invitrogen) were seeded in 6-well plates and treated with 0.1 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma) for 3 h at 37 °C. The medium was removed and the converted dye was solubilized in 100% isopropanol. The absorbance of the converted dye was measured at a wavelength of 570 nm with background subtraction at 670 nm (Synergy HT, BioTek).
10
Genome-wide negative selection screen
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Negative screening was performed using the Decode RNAi Pooled Lentiviral shRNA Screening Libraries: Annotated Genome Negative Selection Kit (Thermo Scientific). Briefly, T-REx HeLa cells (Invitrogen) were infected with a lentiviral siRNA expression library (Thermo Scientific) using TransDux reagent (System Biosciences). Green fluorescent protein-positive cells were selected by puromycin treatment and divided into two populations, one of which was irradiated with 0.75 Gy using a 137Cs irradiator (Gy/min, Best Theratronics); the other served as a non-irradiated control. Four days after irradiation, genomic DNA was purified from each population using a DNA purification kit (Dojindo). Barcode sequences were amplified from 0.5 μg of genomic DNA, purified by gel extraction, and labeled using a Genomic DNA Enzymatic Labeling Kit (Agilent Technologies). After purification using Amicon Ultra-0.5 ml centrifugal filters (Millipore), the labeled barcode sequences were hybridized with microarray slides for 17 h and the slides were then washed according to the Agilent CpG microarray protocol. Cluster analysis of the extracted genes was performed using the information in the GeneCards database (http://www.genecards.org/ ) and KEGG (http://www.genome.jp/kegg/ ).
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