Synchronization of HeLa cells was either done by a single 2 mM thymidine (Sigma-Aldrich, T1895) arrest for 24 h (Figs 1 , 2a,b , 3d , 4a,c , 5c–e,g and 6 and Supplementary Figs 1c, 2b, 3C–E, 4C–D, 5A ) or a double-thymidine arrest (22 h arrest followed after 9 h by a 17 h arrest (Figs 2d , 3a–c and 5a,f and Supplementary Figs 3a, 4a and 5b ). After washout drugs were added: 1 μM Nocodazole (M1404, Sigma-Aldrich), 0.1 μM BI2536 (S1109, Selleck), 10 μM QVD (SML0063, Sigma-Aldrich), 0.5 μM reversine (BML-SC104, Enzo Life Sciences), 2.5 μM (in Fig. 7 ) or 1 μM (in all other Figures) ABT-737 (S1002, Selleck) were added. Dimethyl sulfoxide (D5879, Sigma-Aldrich) was used as solvent control. G2 time points were harvested after 8 h and the first mitotic time point (‘M') 11 h after release from thymidine arrest. All mitotic time points were harvested by shake-off. Doxycycline (D9891, Sigma-Aldrich) was used in the indicated concentrations. Staurosporine (S-9300, LC Laboratories) was used on asynchronous cells for 4 h at 1 μM.
D5879
Manufactured by Merck Group
Sourced in United States, Germany
The D5879 is a laboratory equipment product from Merck Group. It is a precision instrument designed for various scientific and analytical applications. The core function of the D5879 is to provide accurate and reliable measurements, enabling researchers and laboratory professionals to conduct their experiments and analyses effectively.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Nocodazole, by Merck Group (2 mentions)
T8154, by Merck Group (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Doxycycline, by Merck Group (1 mentions)
T1895, by Merck Group (1 mentions)
Abt 737, by Selleck Chemicals (1 mentions)
Bi2536, by Selleck Chemicals (1 mentions)
Staurosporine, by LC Laboratories (1 mentions)
Arium pro, by Sartorius (1 mentions)
Balb c nu nu mice, by Taconic Biosciences (1 mentions)
Bez235, by LC Laboratories (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Milli q, by Merck Group (1 mentions)
Albumax, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
St316, by Beyotime (1 mentions)
Foetal bovine serum (fbs), by Clark Bioscience (1 mentions)
N800425, by Maclin Biochemical Technology (1 mentions)
Prism 8, by GraphPad (1 mentions)
30 protocols using d5879
1
HeLa Cell Synchronization and Drug Treatment
2
Modulation of Signaling Pathways
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The following small molecules were evaluated for induction or reduction of pathway activity. Wnt: CHIR99021(#SML1046) [36 (link)], LY2090314 (#SML1438) [36 (link)], niclosamide (N3510) [37 (link)], LGK-974 (#S7143) [36 (link)]; Hh: SAG dihydrochloride (SAG, #SML1314) [38 (link)], sonidegib (#S2151) [39 (link)], vismodegib (#S1082) [40 (link)]; Notch: suberohydroxamic acid (SBHA, #390585) [41 (link)], DAPT (#S2215) [42 (link)], Dibenzazepine (DBZ, #S2711) [43 (link)], valproic acid sodium salt (VPA, #P4543) [44 (link)]. All reagents were purchased from Selleckchem (Munich, Germany) or Sigma Aldrich. VPA and SAG were dissolved in ultrapure water (Arium pro, Sartorius AG, Göttingen, Germany), and the remaining compounds in DMSO (#D5879, Sigma-Aldrich).
Transfections and evaluations were performed as described above, with the following changes: For induction of the Wnt-pathway in the respective wells, 100 μl conditioned medium (containing wnt3a, see above) was added to the wells four hours after transfection. Compounds were also added four hours after transfection, at a final concentration of 10μM. An equal amount of appropriate solvent (water or DMSO) was added to the solvent control conditions.
Transfections and evaluations were performed as described above, with the following changes: For induction of the Wnt-pathway in the respective wells, 100 μl conditioned medium (containing wnt3a, see above) was added to the wells four hours after transfection. Compounds were also added four hours after transfection, at a final concentration of 10μM. An equal amount of appropriate solvent (water or DMSO) was added to the solvent control conditions.
3
Evaluating BEZ235 Efficacy in Ovarian Cancer
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All experimental procedures involving animals were approved by The National Animal Research Authority (Application ID 4423), and carried out according to the European Convention for the Protection of Vertebrates used for Scientific Purposes.
Female athymic BalbC nu/nu mice (Taconic Tornbjerg, Ejby, Denmark) were inoculated subcutaneously on the hind limb with 5 × 106 cells of either the TOV-21G or TOV-112D cell line in PBS with 1% FBS. After an average time of 3 weeks post inoculation for the TOV-112D model and 4 weeks for the TOV-21G model, the mice were randomised into treatment and control groups. BEZ235 (LC Laboratories, Woburn, MA, USA) was dissolved in dimethyl sulphoxide (DMSO, Sigma-Aldrich D-5879) and then diluted to a final concentration of 6.5 mg ml−1 using polyethylene glycol (PEG300, 20237-1; Sigma-Aldrich). The treatment groups received BEZ235 at a dose of 65 mg kg−1 per day by oral gavage on three consecutive days. This treatment regime was expected to lead to measurable treatment response (e.g., reduced pAKT levels) in responding tumours (Maira et al, 2008 (link); Santiskulvong et al, 2011 (link); Moestue et al, 2013 (link)). The control groups did not receive any treatment. The study design is visualised inFigure 1 .
Female athymic BalbC nu/nu mice (Taconic Tornbjerg, Ejby, Denmark) were inoculated subcutaneously on the hind limb with 5 × 106 cells of either the TOV-21G or TOV-112D cell line in PBS with 1% FBS. After an average time of 3 weeks post inoculation for the TOV-112D model and 4 weeks for the TOV-21G model, the mice were randomised into treatment and control groups. BEZ235 (LC Laboratories, Woburn, MA, USA) was dissolved in dimethyl sulphoxide (DMSO, Sigma-Aldrich D-5879) and then diluted to a final concentration of 6.5 mg ml−1 using polyethylene glycol (PEG300, 20237-1; Sigma-Aldrich). The treatment groups received BEZ235 at a dose of 65 mg kg−1 per day by oral gavage on three consecutive days. This treatment regime was expected to lead to measurable treatment response (e.g., reduced pAKT levels) in responding tumours (Maira et al, 2008 (link); Santiskulvong et al, 2011 (link); Moestue et al, 2013 (link)). The control groups did not receive any treatment. The study design is visualised in
4
Resveratrol Modulates Hippocampal Pathways
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The test agent included resveratrol (R5010, Sigma-Aldrich, St. Louis, MO, USA) [26 (link),86 (link)], a PGC-1α activator, that was microinjected bilaterally and sequentially into the bilateral hippocampal CA3 areas [26 (link)]. The dose of resveratrol used was 100 µmol dissolved in 3% dimethyl sulfoxide (DMSO) (D5879, Sigma-Aldrich) as in our previous reports [26 (link),41 (link)], at a volume of 150 nL on each side. Rats receiving microinjections of DMSO (3%) were used for the volume and vehicle controls. Each rat received only a single pharmacological pretreatment to prevent the confounding effects of drug–drug interactions 30 min before administration of KA (0.5 nmol) or PBS into the left hippocampal CA3 subfield.
5
Medium Preparation for Testicular Toxicity
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To make culture medium, α‐modified Eagle Minimum Essential Medium (αMEM; Gibco: 12000‐022) was dissolved in water purified with Milli-Q (Millipore) at a twofold concentration, and AlbuMAX (Gibco: 11020‐021) was added at 40 mg/mL as a final concentration. Then, 7% NaHCO3 solution was added (0.026 ml/L) to achieve a final concentration of 1.82 g/L. Antibiotic‒antimycotic (15240062, Thermo Fisher Scientific) was added at a 1/100 volume to achieve a final concentration of 100 IU/ml for penicillin, 100 μg/mL for streptomycin, and 250 ng/ml for amphotericin. Lastly, the medium solution was brought to the final volume with MilliQ water. The medium was sterilized with a Millipore filter having a pore size of 0.22 µm and stored at 4 °C. In the testicular toxicity experiment, EE (10006486, Cayman Chemical Company) dissolved in DMSO (D5879, Sigma-Aldrich) was added to the culture medium at the concentrations indicated in the text. In every group, including the control, the amount of DMSO added was 1/10,000 (v/v) of the medium.
6
MTT Assay for Cell Viability
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Cardiomyocytes were seeded into the 96‐well plates at a density of 1 × 104 cells/well, with 8 parallel wells settled. The blank control was settled with culture medium only. Once reached 70% confluence, the cells in each well were cultured with 10 μL 5 mg/mL MTT solution (ST316; Beyotime) in an incubator at 37°C. After 4 hours, the cells in each well were treated with 100 μL dimethyl sulfoxide (DMSO; D5879; Sigma‐Aldrich) for 10 minutes. The OD value was measured at 490 nm using a microplate (MK3, Thermo). The cell viability was calculated as follows: cell viability = (OD value of the experimental well − OD value of the blank well)/OD value of the blank well.
7
Cell Viability Assay with MTT
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After being transfected and cultured for 48 h, the cells in each group were collected and counted. The transfected cells in each group were seeded into the 96-well plate (1 × 104 each well, 8 parallel wells in each group), and the blank control well was set and added to just the culture medium. The cells were then cultured for 24, 48, and 72 h until cells adhered to the well. The cells in each well were then added to 10 μL of MTT (5 mg/mL, ST316, Beyotime) in an incubator at 37°C for 4 h. With the supernatant discarded, the cells in each well were added to 100 μL of DMSO (D5879, Sigma-Aldrich Chemical), followed by oscillation on a table concentrator for 10 min. Subsequently, the OD value was measured at 490 nm by a microplate reader (MK3, Thermo Scientific, Pittsburgh, PA, USA). The experiment was conducted three times to obtain the average value. The cell viability curve was drawn with the time point as abscissa and OD as ordinate.
8
Notch1 Overexpression Sensitivity to Curcumin and CD2066
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Growth susceptibility to the exposure to increasing dosages of curcumin and CD2066 was compared between DND41 cells transduced with a retroviral construct encoding the entire murine Notch1 intracellular fragment CMMP-ICN1-IRES-EGFP (mICN1) and the DND41 counterpart cells transduced with the relative empty control vector CMMP-IRES-EGFP (empty), as previously described [59 (link)]. Cells were seeded in a 24-well flat-bottom plate at 5 × 105 cells/mL and then exposed to the indicated compound concentrations for 48 h. The number of viable cells was analyzed using a Trypan Blue exclusion assay (T8154; Sigma-Aldrich) and normalized to the number of the counterpart cells treated with the vehicle DMSO (D5879; Sigma-Aldrich).
9
Modulation of Alveolar Epithelial A549 Cells
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The alveolar epithelial cells A549 (Dingguo Changsheng) were cultured in RPMI 1640 medium (HyClone) supplemented with 10% foetal bovine serum (Clark) and 1% penicillin/streptomycin in 25 cm2 culture flask at 37°C in 5% CO2. A549 cells were treated by MA with the dosage of 0.1, 0.5, 1 and 5 mmol/L for 6, 12 and 24 hours.24 A549 cells were preincubated with N‐acetylcysteine (NAC; N800425, Macklin) or Resveratrol (Res; R817263, Macklin) dissolved in dimethyl sulfoxide (DMSO; D5879, Sigma) for 1 hour before 5 mmol/L MA stimulation. The concentration of DMSO in the medium never exceeded 0.1% to avoid its toxicity towards the A549 cells. To evaluate the effects of NAC, the cells were divided into control group, 5 mmol/L MA group, 5 mmol/L NAC group, and 5 mmol/L MA plus 5 mmol/L NAC group.25 To evaluate the effects of Res, the cells were divided into control group, 5 mmol/L MA group, 20 μmol/L Res group and three MA plus Res (1, 5, 20 μmol/L) groups.26
10
Cytotoxicity Assay of Compounds
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KOPT-K1, DND41, TALL1 and Loucy cells were seeded in a 24-well flat-bottom plate at 5 × 105 cells/mL, while U937 and THP1 cells were seeded at 2 × 105 cells/mL and then treated at the indicated times and compound concentrations. The number of viable and dead cells was counted by a Trypan Blue exclusion assay (T8154; Sigma-Aldrich) and normalized to the number of cells treated with the solvent DMSO alone (D5879; Sigma-Aldrich). Relative cell viability values were further fitted by a non-linear regression model to calculate the absolute IC50 of the abovementioned compounds for each cell line using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA).
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