Human epidermal keratinocytes (HEKs) (Pansheon Company, China) were maintained for up to five passages with defined keratinocyte serum-free media (Invitrogen, USA). HPV16-positive human cervical carcinoma cell line SiHa and human embryonic kidney cell line 293 T were purchased from the Shanghai Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The latter two cell lines were routinely cultured in Dulbecco's modified Eagle's medium (Hyclone, USA) with 10% heat-inactivated fetal bovine serum (Invitrogen, USA) and 1% penicillin/streptomycin (Invitrogen). All cells were grown in a humidified (37°C, 5% CO2) incubator. Also, miRNA mimics for miR-143 or miR-145, or mimic control, and siRNAs for NRAS or siRNA control, were purchased from RiboBio (China). Nutlin-3a, a p53 activator and MDM2 antagonist, and puromycin were purchased from MedChemExpress Company (China). HEKs or SiHa cells were co-treated with 20 µg ml−1 or 40 µg ml−1 Nutlin-3a for an appropriate time after transfection or transduction.
Nutlin 3a
Manufactured by MedChemExpress
Sourced in United States
Nutlin-3a is a small molecule product offered by MedChemExpress. It is a potent and selective inhibitor of the interaction between the p53 tumor suppressor protein and the E3 ubiquitin-protein ligase MDM2. This interaction is critical for the regulation of p53 activity.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Puromycin, by MedChemExpress (1 mentions)
Recombinant mouse il 15, by Thermo Fisher Scientific (1 mentions)
Thymidine, by Merck Group (1 mentions)
Bn0389, by Biotrend (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Palmitate acid, by Merck Group (1 mentions)
Pifithrin α hydrobromide, by MedChemExpress (1 mentions)
Mk 1775, by MedChemExpress (1 mentions)
Ly2606368, by Selleck Chemicals (1 mentions)
Mi773, by Selleck Chemicals (1 mentions)
Siremadlin, by MedChemExpress (1 mentions)
Idasanutlin, by MedChemExpress (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Senexin a, by Selleck Chemicals (1 mentions)
Thz531, by MedChemExpress (1 mentions)
5 fluorouracil, by Selleck Chemicals (1 mentions)
Triptolide, by Selleck Chemicals (1 mentions)
Pictilisib, by MedChemExpress (1 mentions)
12 protocols using nutlin 3a
1
Investigating miRNA-mediated regulation in cancer cells
2
Apoptosis Induction in Mouse NK Cells
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Around 2 × 10 4 mouse NK cells were cultured in CR-10 media with 5 ng ml -1 recombinant mouse IL-15 (PeproTech; 210-15) and control DMSO or 2.5 μM Nutlin-3a (MedChem Express, HY-10029) for 24 h before intracellular staining for cleaved caspase 3 by flow cytometry.
3
Synchronized Ciliogenesis in RPE1 Cells
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The following compounds were used: ZM447439 2 μM (Selleck Chemicals, S1103), dihydrocytochalasin‐B 4 μM (DHCB, Sigma‐Aldrich, D1641), Camptothecin (CPT, APExBIO, A2877) and Nutlin‐3a (MedChemExpress, HY‐10029). To all untreated controls, solvent only was administered. To induce ciliogenesis, RPE1 cells were seeded on glass coverslips in 6‐well plates. After 24 h, cells were washed with PBS and exposed to serum‐free medium for another 48 h before fixation. Synchronization of RPE1 cells in Fig 7 was performed by arresting cells with 2 mM thymidine (Sigma‐Aldrich, T1895) for 24 h, followed by release in fresh medium containing nocodazole 200 nM (BioTrend, BN0389) for 14 h. Mitotic cells were then harvested by selective shake‐off, washed four times and released into fresh medium. To dissociate centrosome clusters in telophase, nocodazole 1 μM was added 1.5 h after the release.
4
Modulation of AML12 Hepatocyte Metabolism
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AML12 cells were obtained from Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM/F‐12 supplemented with 10% foetal bovine serum (FBS) (Gibco), 1% ITS (Gibco), 40 ng/mL Dexamethasone (Sigma‐Aldrich) and incubated in a humidified incubator at 37°C and 5% CO2. AML12 cells were cultured in the medium without the supplementation of insulin at the beginning of all treatment. The cells at 70% confluence were exposed to palmitate acid (Sigma‐Aldrich), nutlin‐3a or pifithrin‐α hydrobromide (Med Chem Express, USA) for 24 hours. For the insulin stimulation experiments, the cells were treated with or without 100 nmol/L insulin for 20 minutes.
5
Inhibitors for Cancer Research
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6
Cell Viability Assay with CCK8
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Cell viability was detected by CCK8 assay (C6030, New Cell & Molecular Biotech Co., Ltd). Briefly, cells were seeded in 96-well plates with a density of 2 × 103 cells/well. After treatments, CCK8 was added to the cell culture medium and maintained for additional 1 h. The absorbance was determined at 450 nm by a microplate reader (BioTek). Nutlin-3a (HY-10029), Siremadlin (HY-18658), Idasanutlin (HY-15676), and HY-100692 were purchased from MCE MedChemExpress. MI773 (S7649) was purchased from Selleck.
7
Evaluation of Anti-Cancer Compounds
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8
Evaluating Pharmacological Inhibitors
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NVP-2, Nutlin-3a, 5-fluorouridine, 5-fluorodeoxyuridine, THZ531 and Pictilisib were from MedChemExpress. 5-fluorouracil, Senexin A, OTS964 and Triptolide were from Selleck Chemicals. YLK-5–124 and THAL-SNS-032 were a gift from Nathanael S. Gray’s Laboratory (Stanford University, USA). iCDK9 was a gift from Qiang Zhou’s Laboratory (University of California, Berkley, USA). PP2A activator DT-061 was a gift from Jukka Westermarck’s Laboratory (University of Turku, Finland).
9
High-Throughput Organoid Drug Screening
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Organoids were seeded in 96-well plates on day 0 at an initial seeding density of 1000 cells per well. Rucaparib (S1098, Selleck, Houston, TX, USA), niraparib (S2741, Selleck, Houston, TX, USA), olaparib (S1060, Selleck, Houston, TX, USA), carboplatin (S1215, Selleck, Houston, TX, USA), nutlin3a (HY-10029, MedChem Express, Monmouth Junction, NJ, USA), and SNS-032 (S1145, Selleck, Houston, TX, USA) were added on day 7 at the indicated concentrations. The culture medium was refreshed, and a new drug dose was introduced every 3.5 days. The cell viability was assessed on day 14 by using a CCK8 assay kit (K1018, APExBIO, Houston, TX, USA) with incubation at 37 °C for 4 h. Measurements were performed using a microplate reader (PerkinElmer, Waltham, MA, USA), and the results were normalized to DMSO controls.
10
Inhibition of Cell Proliferation
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The reagent MG132 (HY‐13259, Med Chem Express, Shanghai, China) was used at 10 μm . The reagent Nutlin‐3a (HY‐10029, Med Chem Express) was used at 10 μm in vitro (IC50 = 17.75 and 25.67 µm for SW 1990 and HPAC, respectively, Fig. S1 ) and 100 mg·kg−1in vivo. Cycloheximide (HY‐12320, Med Chem Express) was used at 1 μm .
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