IMR90 cells from PD 25–35 were grown in DMEM media with 10% FBS under 3% oxygen and 5% carbon dioxide. Nutlin-3a (Sigma, SML0580–5MG) was dissolved in DMSO to a 5mM stock solution and added to IMR90 cells to a final concentration of 5uM. Treatment of IMR90 cells with Nutlin-3a resulted in increased p53 protein levels and induction of p21 expression as has been previously described (Vassilev et al., 2004 (link)) (Figure S1A ). Treatment with Nutlin-3a also led to strong accumulation of p53 in the nucleus, and consistent with previous reports (Choi et al., 2019 (link)), this includes locations proximal to nuclear speckles as identified by the speckle-marking antibody, ab11826 (Figure S1B ; (Ilık et al., 2020 )). Additionally, ChIP-qPCR shows that p53 binds to its consensus sites at the p21 enhancer and promoter throughout 2, 6, and 9 hours of Nutlin-3a treatment (Figures S1C and S1D ), demonstrating persistent interaction of p53 with its p21 binding sites throughout the time points examined.
Nutlin 3a
Manufactured by Merck Group
Sourced in United States, Germany
Nutlin-3a is a chemical compound developed by Merck Group. It is a potent and selective inhibitor of the interaction between the p53 tumor suppressor protein and the MDM2 (murine double minute 2) oncoprotein. Nutlin-3a is primarily used as a research tool in cellular and biochemical studies to investigate the p53-MDM2 pathway and its role in various cellular processes.
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Lab products found in correlation
Etoposide, by Merck Group (8 mentions)
Cycloheximide (chx), by Merck Group (7 mentions)
Doxorubicin, by Merck Group (6 mentions)
Mg132, by Merck Group (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Doxorubicin, by Cayman Chemical (5 mentions)
Cisplatin, by Merck Group (4 mentions)
Dmem f12 media, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Doxycycline, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (3 mentions)
Rpe 1 htert cells, by American Type Culture Collection (3 mentions)
Actinomycin d, by Cayman Chemical (3 mentions)
Dmem with pyruvate, by Thermo Fisher Scientific (3 mentions)
2 mercaptoethanol, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Trichostatin a, by Merck Group (3 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
90 protocols using nutlin 3a
1
Nutlin-3a Induces p53-Dependent p21 Expression
2
Modulated Electro-Hyperthermia for Cell Viability
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Cells grown on poly-L-lysine (Sigma-Aldrich) coated coverslips were treated with mEHT for 60 min at 42 °C between two plane-parallel electric condenser plates using a Lab-EHY 100 device (Oncotherm Kft, Budaors, Hungary). After the treatment, coverslip cultures were put into fresh culture medium until further processing. When mEHT was combined with nutlin-3a (Sigma-Aldrich), cells were treated with mEHT for 60 min at 42 °C and then placed in complete medium containing 10 µM nutlin-3a. Twenty-four hours post-treatment, resazurin (Sigma-Aldrich) viability assay was performed. Experiments were repeated at least three times.
3
Preparation of Inhibitor Stock Solutions
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The CRM1 antagonist, termed a selective inhibitor of nuclear export (SINE), KPT-330 (Selleck Chemicals, Houston, TX) and imatinib (Santa Cruz Biotechnology, Dallas, TX) were dissolved in DMSO at 50 mM for KPT-330 and 100 mM for imatinib. The selective casein kinase 2 (CK2) inhibitor tetrabromobenzotriazole (TBB) (Abcam, Cambridge, MA) and the MDM2 antagonist Nutlin-3a (Sigma-Aldrich, St. Louis, MO) were dissolved in DMSO at 100 mM for TBB and 30 mM for Nutlin-3a. These solutions were then aliquoted and stored at −80°C.
4
Nutlin-3a Inhibitor Mechanism
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5
Rapid Protein Depletion Assay
6
Co-culture of B-ALL PDX with MSCs
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For co-culture experiments with Luciferase-expressing B-ALL PDX samples, 3x104/cm2 mesenchymal stem cells (MSC) were plated in 96-well tissue culture plates and 2x104 B-ALL PDX cells were added in StemSpan Serum-Free Expansion Medium II (SFEM II, STEMCELL Technologies, Cambridge, UK).17 (link),18 (link) Cells were treated for 5 days with indicated drugs and luminescence analyzed with the Steady-Glo Luciferase Assay System (Promega, Southampton, UK), according to manufacturer’s instruction, and detected with Infinite m200 Pro microplate reader (Tecan, Reading, UK). The following reagents and inhibitors were used, S3I-201 (Cayman Chemical, MI, USA), Napabucasin (BBI608; MedChemExpress, NJ, USA), C188-9 (MedChemExpress and Adooq Biosciences, CA, USA), Nutlin-3a (Merck Life Science, Dorset, UK) and Idasanutlin (Bio-Techne, Abingdon, UK).
7
Cell Culture Conditions for Cancer Lines
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All cell lines were obtained from the
American Tissue Collection Center (ATCC) and
cultured in high-glucose Dulbecco’s Modified Eagle’s
Medium (HCT116, MDA-MB-231) or Roswell Park Memorial Institute medium 1640
medium (H460) supplemented with 10% fetal bovine serum,
100 IU ml−1penicillin,
100 μg ml−1streptomycin and
0.25 μg ml−1amphotericin
B (Life Technologies)
at 37 °C with 5% CO2. Transduced cell lines were
maintained in
0.5–2.0 μg ml−1puromycin after selection was
completed. For induction of dox-regulated vectors, cell culture medium was supplemented
with
1–2 μg ml−1doxycycline (Sigma).
HCT116 cells were treated with 10 μM nutlin-3a (Merck).
CDDP was used at
0.5 μg ml−1,
5-fluorouracil at
375 μM.
American Tissue Collection Center (ATCC) and
cultured in high-glucose Dulbecco’s Modified Eagle’s
Medium (HCT116, MDA-MB-231) or Roswell Park Memorial Institute medium 1640
medium (H460) supplemented with 10% fetal bovine serum,
100 IU ml−1penicillin,
100 μg ml−1streptomycin and
0.25 μg ml−1amphotericin
B (Life Technologies)
at 37 °C with 5% CO2. Transduced cell lines were
maintained in
0.5–2.0 μg ml−1puromycin after selection was
completed. For induction of dox-regulated vectors, cell culture medium was supplemented
with
1–2 μg ml−1doxycycline (Sigma).
HCT116 cells were treated with 10 μM nutlin-3a (Merck).
CDDP was used at
0.5 μg ml−1,
5-fluorouracil at
375 μM.
8
Genotoxic Stress Induction in Neuroblastoma Cell Lines
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To induce genotoxic stress, NB cell lines were treated with various concentrations of the genotoxic drugs (as indicated in the figures)nutlin-3a (SML0580; Merck), doxorubicin (D1515; Merck), etoposide (E1383; Merck), cisplatin (C2210000; Merck), and JNJ-26854165 (S1172; Selleckchem) and also treated with respective control (adding DMSO to media for nutlin-3a, etoposide, and JNJ treatment, adding PBS to media for cisplatin and adding media only for DOX treatment in a corresponding volumes not exceeding 1%). We harvested cells 24 hours after drug treatment for further analysis. SH-SY5Y treated with leptomycin B (LMB; Merck L2913) at a concentration of 5 nmol/L (2.5 ng/mL) for 4 hours followed by nutlin-3a treatment in two different doses 2.5 and 5 mmol/L and then harvested after 20 hours for immunostaining. SH-SY5Y and IMR-32 cells were also treated with selinexor (S7252, Selleckchem) alone at 50 nmol/L concentration or in combination with nutlin-3a (doses indicated in figure) for 24 hours and then harvested for further analysis. To perform drug synergy assessment, NB cells were plated in 96-well plate and then treated with single or combinations of compounds and subsequently analyzed for cell viability 48 hours posttreatment. To check the synergy between the drugs, Chou-Talalay combination index was calculated using Com-puSyn software (31) .
9
Endometrial and Ovarian Cancer Cell Culture
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The endometrial carcinoma cell line ECC1 was maintained in DMEM/F12 medium. HEC1B, SPAC1L (both EC) and the ovarian carcinoma cell line OVMz were cultivated in RPMI-1640 medium (PAA Laboratories, Pasching, Austria). Media were supplemented with 10% fetal bovine serum.
Antibodies to the ectodomain of L1CAM (monoclonal antibody (mAb) L1-11A, a subclone of UJ127.11) were described before [30 (link)];[31 (link)]. The antibodies for detection in Western blot against GAPDH and p53 (DO-1) were from SantaCruz Biotechnology (Heidelberg, Germany). 5′-AzaC, TSA and Nutlin-3a were obtained for Sigma-Aldrich (St. Louis, USA) and dissolved in serum free medium or DMSO.
Antibodies to the ectodomain of L1CAM (monoclonal antibody (mAb) L1-11A, a subclone of UJ127.11) were described before [30 (link)];[31 (link)]. The antibodies for detection in Western blot against GAPDH and p53 (DO-1) were from SantaCruz Biotechnology (Heidelberg, Germany). 5′-AzaC, TSA and Nutlin-3a were obtained for Sigma-Aldrich (St. Louis, USA) and dissolved in serum free medium or DMSO.
10
Inhibition of HSV DNA Replication
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The HSV DNA replication inhibitor acyclovir (Sigma) was used to supplement the postinfection low-serum medium to a final concentration of 200 μM. Nutlin-3a (Sigma) was used to supplement low-serum medium to the concentrations indicated in Fig. S3. Control treatments contained only medium (untreated) or a volume of dimethyl sulfoxide (DMSO) equivalent to that of the highest concentration of Nutlin-3a used. HFFs were pretreated with the concentrations of Nutlin-3a indicated in Fig. S3 for 1 h. Infections were carried out normally, without the addition of Nutlin-3a. Following removal of the inoculum after absorption, the low-serum medium added back contained the concentration of Nutlin-3a used in the pretreatment step. Cells were incubated in low-serum medium supplemented with etoposide (Sigma) at a final concentration of 20 μM for the periods of time indicated in Fig. 2D and Fig. 3D .
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