For mTOR perturbation experiments, cells were transiently transfected with the RPS2-BE fusions as described above (see transient transfection section), and expression of the constructs was induced via the addition of doxycycline at a final concentration of 2 μg/mL and incubated at 37 °C for 24 hrs. After, Torin-1 (Cell Signaling, Cat # 14379) dissolved in dimethyl sulfoxide (DMSO) was added to pre-warmed DMEM + 10% FBS, adding the media to the cultured cells yielded a final concentration of 100 nM. A set of cells were treated with a DMSO vehicle lacking Torin-1 to serve as a control. The cells were then incubated at 37 °C for 48 hrs, after which the media was aspirated, and cells were resuspended in 300 μL of TRIzol followed by RNA extraction, library prep, and sequencing (see above). To ensure the reliability of the results, the experiments were carried out on three replicates.
Torin 1
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Torin-1 is a small molecule that functions as a potent and selective inhibitor of the mammalian target of rapamycin (mTOR) kinase. mTOR is a central regulator of cell growth, proliferation, and metabolism. Torin-1 inhibits both mTORC1 and mTORC2 complexes, making it a useful tool for studying mTOR signaling pathways.
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Lab products found in correlation
Rapamycin, by Merck Group (4 mentions)
Bafilomycin a1, by Merck Group (3 mentions)
Rapamycin, by Cell Signaling Technology (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Kapa rna hyperprep kit with riboerase, by Roche (2 mentions)
Fugene hd, by Promega (2 mentions)
Truseq stranded mrna library prep, by Illumina (2 mentions)
Direct zol miniprep, by Zymo Research (2 mentions)
Wortmannin, by Merck Group (2 mentions)
Celltrace calcein violet, by Thermo Fisher Scientific (2 mentions)
Mouse catalog i9646, by Merck Group (2 mentions)
Human catalog i1395 il 6, by Merck Group (2 mentions)
Propranolol catalog p0884, by Merck Group (2 mentions)
Isoproterenol catalog i6504, by Merck Group (2 mentions)
Propranolol, by Bio-Techne (2 mentions)
Chloroquine, by Enzo Life Sciences (2 mentions)
Isoproterenol, by Bio-Techne (2 mentions)
Cyto id autophagy detection kit 2, by Enzo Life Sciences (2 mentions)
Etomoxir, by Bio-Techne (2 mentions)
Mouse catalog 713704 and human catalog 713604 gm csf, by BioLegend (2 mentions)
35 protocols using torin 1
1
Investigating mTOR Perturbation Effects
2
Torin-1 Treatment of HEK293T Cells
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HEK293T cells were treated at a final concentration of 250 nM Torin-1 (Cell Signaling Technology, #14379) in standard HEK293T media for 18 hours prior to UV-crosslinking and harvesting.
3
Evaluating Curcumin's Cellular Effects
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4
Inducible STAMP Fusion Protein Expression
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For stable cell STAMP fusion protein expression cells were induced with 50ng/ml (low) or 1μg/ml (high) doxycycline in DMEM for 24–72 hours, followed by Trizol extraction and Direct-zol miniprep (Zymo Research) column purification in accordance with manufacturer protocol. Uninduced cells of the same genetic background were used as negative controls. For transient transfections, ~1 million cells were transfected with 2μg expression construct using Fugene HD (Promega) according to manufacterer’s protocol. Upon Agilent TapeStation quantification, 500ng RNA was used as input material to make total RNA-seq libraries with either TruSeq Stranded mRNA Library Prep (Illumina) or KAPA RNA HyperPrep Kit with RiboErase (Roche) following the provided protocols. For mTOR perturbation experiments, cells were treated with 100nM Torin-1 (Cell Signaling) or DMSO vehicle control alongside 1μg/ml doxycycline induction and harvested for RNA after 72 hours 37C incubation.
5
Isolation and Cultivation of Chick Myoblasts
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Myoblasts were isolated from the thigh muscles of 13-day-old chick embryos (Nakashima et al., 1998 (link)). Briefly, the muscle tissue obtained from the embryo was digested with dispase (Gibco, USA), and the cell suspension was transferred to an uncoated culture dish to allow for fibroblast attachment. Cell numbers were counted, and the cells were then plated onto gelatin-coated 6-well plates (Iwaki SciTech, Japan) at a density of 2.0 × 105 cells/well. The chicken embryo extract was prepared from 10-day chick embryos. The embryos were rinsed with phosphate-buffered saline (PBS), passed through a 50 mL syringe, and an equal volume of PBS was added. The supernatant collected by centrifugation (10,000 rpm for 1 h) at 4°C was used as the chicken embryo extract. Chick myoblasts were cultured in M-199 medium containing 15% calf serum and 2.5% chicken embryo extract and were grown at 37°C in a humidified atmosphere of 5% CO2 for 7 days. On day 7, the cells had formed myotubes and were incubated for 3h in serum-free M-199 medium containing Torin1 (100 nM, Cell Signaling Technology, USA). Torin1 is an ATP-competitive inhibitor that targets the kinase domain of mTOR (Thoreen et al., 2009 (link)).
6
FGF Signaling and Intracellular Trafficking
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FGF ligands (PeproTech) were used at 50 ng/ml for 4 h or overnight. c‐Jun N‐terminal kinase (JNK) inhibitor (SP600125, Sigma‐Aldrich, Milan, Italy) was used at 50 μM for 12 h. Bafilomycin A1 (Sigma‐Aldrich) was used at 100 nM for 3–4 h. Torin 1 (Cell Signaling) was used at 1 μM for 2 h. Concanamycin A (Sigma‐Aldrich, Milan, Italy) was used at 100 nM for 1 h. Proteasomal inhibitor (MG132, Sigma‐Aldrich, Milan, Italy) was used at 10 μM for 6 h. Actinomycin (Sigma‐Aldrich) was used at 1 μg/ml for 4 h. ER‐Tracker BODIPY Green (BODIPY™ FL Glibenclamide, for live‐cell imaging, Thermo Fisher) was used at 1 μM for 30 min at 37°C in DMEM (w/o supplements) in dark and then fixed with 4% PFA for 2 min at 37°C.
7
Rapamycin and Torin-1 Inhibitor Assay
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Rapamycin (RAPA, #9904, Cell Signaling Technology, Danvers, MA, USA) and Torin-1 (#14379, Cell Signaling Technology) inhibitors were resuspended in DMSO, diluted in complete RPMI, and for all treatments added to cell suspensions at a final concentration of 100 nM24 (link).
8
Ferroptosis Induction and Inhibition Assay
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Rapamycin was purchased from Thermo Fischer Scientific (Cat#FSBBP2963-1). Torin 1 was purchased from Cell Signalling Technologies (Cat#14379S). RSL3 was purchased from Jomar Bioscience (Cat# S8155). Erastin (Cat#E7881), ferric ammonium citrate (FAC) (Cat#F5879), deferoxamine mesylate salt (Dfo) (Cat#D9533), deferiprone (Dfp) (Cat#379409), bafilomycin A1 (Baf) (Cat# B1793) and MG-132 (ready made solution) (Cat# M7449) were purchased from Sigma Aldrich. All other reagents were purchased from Sigma Aldrich unless otherwise stated.
9
Rapamycin and Torin 1 Inhibition Assay
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Rapamycin was from Calbiochem (53123-88-9) and was prepared as 100 μM stock in DMSO. Torin 1 was purchased from Cell Signaling Technology (14379) and was dissolved as 100 μM stock in DMSO. Calf intestinal alkaline phosphatase (CIP) was from New England Biolabs (M0290S). Hexadimethrine bromide (Polybrene) was from Sigma-Aldrich (107689). Antibodies used were: anti-LARP6 antibody from Abnova (H00055323-B01P), anti-HA antibody from Sigma-Aldrich (H9658), anti-phospho AKT (S473), anti-pan AKT, anti-phospho S6K (T389), and anti-S6K antibodies from Cell Signaling Technology (12694, 4691, 9205, and 2708, respectively), anti-collagen α1(I) antibody from Rockland (600-401-103), anti-collagen α2(I) antibody from Santa Cruz Biotechnology (sc-8786), anti-calnexin antibody, human anti-fibronectin antibody and anti-STRAP antibody from BD Transduction Laboratories (610523, 610077, and 611346, respectively), rat anti-fibronectin from Millipore (AB1954), anti-β-actin antibody from Abnova (ab8227), anti-Sec61β antibody from Thermo Scientific (PA3-015) and anti-raptor antibody from Bethyl Laboratories (A300-553A-M).
10
VHL-deficient Kidney Cancer Cell Protocols
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VHL-defective kidney cancer cell lines, RCC4 and 786-O, were from Cell Services at the Francis Crick Institute and were maintained in DMEM (high glucose, GlutaMAX Supplement, HEPES, Thermo Fisher Scientific, no. 32430100) with 1 mM sodium pyruvate (Thermo Fisher Scientific, 12539059) and 10% FBS at 37 ˚C in 5% CO2. Cells were confirmed to be of the correct identity by STR profiling and to be free from mycoplasma contamination.
Hypoxic incubation was performed using an InvivO2 workstation (Baker Ruskinn) in 1% O2 and 5% CO2 for 24 hours. To inhibit mTOR, cells were treated with 250 nM of Torin 1 (Cell Signaling Technology, no. 14379) for 2 hours.
An overview of the experimental interventions and analyses is provided in Supplementary Data1 . Biological replicates are individual experiments using different clones derived from the same cell line. All other replicates are defined as technical replicates.
Hypoxic incubation was performed using an InvivO2 workstation (Baker Ruskinn) in 1% O2 and 5% CO2 for 24 hours. To inhibit mTOR, cells were treated with 250 nM of Torin 1 (Cell Signaling Technology, no. 14379) for 2 hours.
An overview of the experimental interventions and analyses is provided in Supplementary Data
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