Breast cancer cell lines MDA-MB-231 (ATCC #HTB-26, RRID:CVCL_0062), MDA-MB-468 (ATCC #HTB-130, RRID:CVCL_0419), MCF7 (ATCC #HTB-22, RRID:CVCL_0031) and HCC70 (ATCC #CRL-2315, RRID:CVCL_1270) were cultured at 37°C and 5% CO2 in DMEM (Gibco #41965039) supplemented with 10% fetal bovine serum (FBS; Gibco #10270-106). Because of many serial passages, MCF7 cell authenticity was confirmed by Microsynth AG. Other cell lines were freshly obtained from American Type Culture Collection (ATCC). The Ba/F3 pro-B cell line (DSMZ #ACC-300, RRID:CVCL_0161) was obtained from Leibniz-Institute DSMZ and grown in RPMI-1640 (Gibco #21875091) supplemented with 10% FBS and 10 ng/ml interleukin 3 (IL-3; Miltenyi). CRISPR-Cas9 engineering of Ba/F3 cells is described in the supplementary information. All cell lines were tested for Mycoplasma with the MycoAlert Mycoplasma Detection Kit (Lonza #LT07-418) before use and were cultured for maximum 49 days after thawing. Serine depleted media corresponds to DMEM without serine and glycine (US Biological Life Sciences #D9802-01), supplemented with 4.5 g/l glucose, 3.7 g/l sodium bicarbonate, 1:100 glutamax and 10% dialyzed serum (Gibco #A3382001).
A3382001
Manufactured by Thermo Fisher Scientific
The A3382001 is a laboratory equipment product from Thermo Fisher Scientific. It is designed to perform a core function within the laboratory setting. No further details or interpretations about the intended use of this product are provided.
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Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Miltenyi Biotec (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
L arginine, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hbss medium, by Thermo Fisher Scientific (1 mentions)
Transit 2020, by Mirus Bio (1 mentions)
Rotenone, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Anti cd14 pacific blue clone 63d3, by BioLegend (1 mentions)
Bms 303141, by Bio-Techne (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
G7021, by Merck Group (1 mentions)
Tlrl eblps, by InvivoGen (1 mentions)
Tlrl eklps, by InvivoGen (1 mentions)
P2636, by Merck Group (1 mentions)
9 protocols using a3382001
1
Cultured Breast Cancer Cell Lines
2
Polysome Profiling of HEK293T Cells
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The polysome profiling protocol employed is previously described (57 (link)). Briefly, HEK293T cells were seeded into four 15 cm2 culture dishes per condition and maintained in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), penicillin (50 μg/ml), and streptomycin (50 μg/ml) in a 37 °C incubator until they reached 80% confluency on the day of the experiment. DMEM without leucine (226–024; Crystalgen) or control DMEM (226–033; Crystalgen) were combined with 10% dialyzed FBS (A3382001; Gibco) for amino acid deprivation experiments.
3
BRET Assay for Gγ-GFP2 and Gα-RLuc8 Interactions
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HEK293T (ATCC; CRL-11268) cells overexpressing human Gγ8-or Gγ9-GFP2 and wildtype or mutant human Gαo-RLuc8 donors were used for BRET assays. Cells were grown in Dulbecco’s modified Eagle’s medium (Corning; 10-017-CV) + 10% fetal bovine serum (Gibco; A3382001) + 1% penicillin/streptomycin at 37°C and 5% CO2.
4
Transient tRNA transfection in mammalian cells
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HEK293T and MEF cells were grown in Dulbecco’s modified Eagle’s medium, with GlutaMAXTM supplement (DMEM + GlutaMAX, Gibco) with 10% fetal bovine serum and 1% Penicillin–Streptomycin at 37 °C, with 5% CO2. Then tRNA transfections were performed in 6-well plates. In vitro transcribed tRNA was mixed with the 4 µg of Arg sensor plasmid, and this mixture was directly added to the cells in combination with Lipofectamine 2000. For Figure 3 , right, 1200 ng tRNA was transfected. For Figure 4 , we used dialyzed serum (A33820-01, Gibco) and DMEM media deficient in both L-lysine and L-arginine (88364, Thermo Scientific). L-lysine was added to make the 146 mg/L final concentration. After 48 h, the transfected cells were washed once with phosphate-buffered saline (PBS, Corning) and harvested by scraping and centrifugation. Cell pellets were lysed in 4 × SDS Sample Buffer at the w:v ratio of 1:20 (1 mg cell pellet: 20 µL buffer), followed by boiling the samples for 10 min. Then 10 µL of each sample was loaded for SDS–PAGE electrophoresis.
5
Cell Starvation and Transfection Protocols
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Cell culture and transfection For starvation, cells were washed with PBS twice and cultured in FBS-free DMEM or Hanks' Balanced Salt Solution (HBSS) medium (Invitrogen). For glucose starvation, cells were washed with PBS and cultured in DMEM without glucose (GIBCO#11966025) supplemented with 10% FBS and sodium pyruvate. For amino acids starvation, cells were washed with PBS and cultured in DMEM supplemented with 10% dialyzed FBS (GIBCO#A3382001). Cell transfection was performed with TransIT 2020 (for pDNA; Mirus) or Lipofectamine 2000 reagents (for siRNA; Invitrogen) in GIBCOOpti-MEM I Reduced Medium (GIBCO#11524456) using the manufacturers' protocols. Final siRNA concentrations of 20-50 nM was used for silencing for 3-5 days.
6
Monocyte Metabolism Modulation Techniques
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Peripheral blood was collected with informed written consent and ethical approval was obtained from Wales Research Ethics Committee 6 (13/WA/0190). Mononuclear cells were obtained by density gradient centrifugation (Histopaque-1077 (10771), Merck) Human CD14+ monocytes were isolated using CD14 microbeads (130-050-201; Miltenyi Biotec). The purity of monocytes was routinely monitored using anti-CD14 Pacific Blue (clone 63D3; 367122; BioLegend) via flow cytometry.
Monocytes (1.0 × 106/mL) were activated with LPS (10 ng/mL; Ultrapure, tlrl-eblps; Invivogen) and cultured in glucose (G7021) or fructose (F3510; 11.1 mM; Merck) containing glucose-free RPMI (11879020; Thermo Fisher) supplemented with 10% dialysed foetal bovine serum (FBS; A3382001; Thermo Fisher Scientific). 2-DG (0.1–1 mM; D8375), oligomycin (1 µM; 75351), antimycin A (1 µM; A8674) and rotenone (1 µM; R8875) were obtained from Merck. The ACLY inhibitor, BMS303141 (4609), was purchased from Tocris. LPS purchased from Invivogen (Escherichia coli K12; tlrl-eklps) was used for the varying glucose concentration experiments.
Cells were harvested and analysed for flow cytometry (acquired with NovoExpress V1.4.1) and supernatants were stored at −20 °C for cytokine analysis.
Monocytes (1.0 × 106/mL) were activated with LPS (10 ng/mL; Ultrapure, tlrl-eblps; Invivogen) and cultured in glucose (G7021) or fructose (F3510; 11.1 mM; Merck) containing glucose-free RPMI (11879020; Thermo Fisher) supplemented with 10% dialysed foetal bovine serum (FBS; A3382001; Thermo Fisher Scientific). 2-DG (0.1–1 mM; D8375), oligomycin (1 µM; 75351), antimycin A (1 µM; A8674) and rotenone (1 µM; R8875) were obtained from Merck. The ACLY inhibitor, BMS303141 (4609), was purchased from Tocris. LPS purchased from Invivogen (Escherichia coli K12; tlrl-eklps) was used for the varying glucose concentration experiments.
Cells were harvested and analysed for flow cytometry (acquired with NovoExpress V1.4.1) and supernatants were stored at −20 °C for cytokine analysis.
7
Cell Viability Assay for Cytotoxic Effects
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Cell viability was
measured using the nuclei count data (NucBlue staining) in nontransfected
HTLA cells. We defined cytotoxic effects as a 20% reduction of cell
viability compared to the controls. Briefly, cells were seeded at
a density of 10 000 cells well–1 in white, optical
bottom 384-well plates (Sigma-Aldrich, CLS3765) coated with 0.1 mg
mL–1 poly-l -lysine (Sigma-Aldrich, P2636).
The following day, cells were starved for 1 h in starving media (DMEM
supplemented with 1% dialyzed fetal bovine serum (dFBS, Thermo Fisher,
A3382001), 1× penicillin/streptomycin) before addition of BPA,
DEP, TPP, plastic extracts, and the three mixes. The exposure lasted
23 h before being stained with NucBlue and imaged as described above.
To confirm that cell viability was similar between transfected cells,
we compared nontransfected cells to those expressing MTNR1B and AVPR2.
No differences were observed at concentrations relevant to the screen
(Figure S4 ), and results were generalized
to all receptors.
measured using the nuclei count data (NucBlue staining) in nontransfected
HTLA cells. We defined cytotoxic effects as a 20% reduction of cell
viability compared to the controls. Briefly, cells were seeded at
a density of 10 000 cells well–1 in white, optical
bottom 384-well plates (Sigma-Aldrich, CLS3765) coated with 0.1 mg
mL–1 poly-
The following day, cells were starved for 1 h in starving media (DMEM
supplemented with 1% dialyzed fetal bovine serum (dFBS, Thermo Fisher,
A3382001), 1× penicillin/streptomycin) before addition of BPA,
DEP, TPP, plastic extracts, and the three mixes. The exposure lasted
23 h before being stained with NucBlue and imaged as described above.
To confirm that cell viability was similar between transfected cells,
we compared nontransfected cells to those expressing MTNR1B and AVPR2.
No differences were observed at concentrations relevant to the screen
(
to all receptors.
8
Culturing Human Osteosarcoma Cells (U2OS)
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Human osteosarcoma cells (U2OS) (https://www.cellosaurus.org/CVCL_0042 ) were originally obtained from the American Tissue Culture Collection (ATCC® HTB-96™) and were maintained at the Heinrich Pette Institute for several years52 (link). The cells were used in previous studies. U2OS cells were cultured in full growth medium composed of DMEM low glucose (SIGMA, D5546), supplemented with 10% FCS (REF), 1% penicillin/streptomycin (SIGMA, P4333), 100 µM pyruvate (SIGMA, S8636), 4 mM L-glutamine solution (SIGMA, G7513-100ML), and 1 g/L D-( +)-glucose solution (SIGMA, G8769). The cells were cultured in T75 flasks (Greiner, 658,175) at 37 °C with 5% CO2. During the cultivation process, the growth medium was refreshed every other day, and cells were reseeded once they reached approximately 80% confluence. For experiments involving reduced glucose or glutamine levels, glutamine-only or glucose-only medium conditions were prepared accordingly. Additionally, experiments were conducted using either default or dialyzed FCS (Thermo-Fischer, A3382001).
9
Metabolic Profiling of EC Cells
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EC cells cultured for 24 h in DMEM or DMEM:F12 with 10% FBS with 1 mM sodium pyruvate without glucose were applied to the Pyruvate Dehydrogenase Activity Colorimetric Assay (K609-100; BioVision), Lactate Dehydrogenase Activity Colorimetric Assay (K726-500, BioVision), and ADP/ATP Ratio Assay (MAK135, Sigma–Aldrich) according to the manufacturers’ instructions. EC cell lysates and conditioned medium after culturing for 12 h in DMEM or DMEM:F12 with 1 mM sodium pyruvate without glucose and with dialyzed FBS (A33820-01; Thermo Fisher Scientific) were applied to the Lactate-Glo Assay (J5021; Promega) according to the manufacturer’s instruction. PDH and LDH activity values were corrected by cell numbers and lactate detection values were corrected by values from the CellTiter 96 AQuous One Solution Proliferation Assay (Promega).
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