Six-week-old female NOD/SCID mice were purchased from Charles River and housed with food and water ad libitum, five animals were kept in each cage. A549 LIMD1−/− and LIMD1+/+ were grown until reaching the beginning of the exponential growth phase and then detached and resuspended in Matrigel 5 mg/mL (Sigma). First, 1 × 106 cells in 100 µL of Matrigel were injected subcutaneously into the mice, 20 with A549 LIMD1−/− and 20 with A549 LIMD+/+. Tumour growth was measured three times per week using callipers and the tumour size calculated using the formula V = (length2 × width)/2. Once the average tumour size was between 150 and 200 mm3, each group was randomised into two groups to maintain equal tumour size between groups and once a week they received either PF-477736 (7.5 mg/kg per dose) or vehicle (50 nM sodium acetate and 4% dextrose, pH 4 (Sigma)) both dosed twice a day with 6 h difference. Tumour size and mice weight were monitored three times per week and the experiment was stopped when the tumour size exceeded 1.44 cm3. Mice were then culled and the tumours harvested, sectioned longitudinally and each section was fixed in 10% neutral buffered formalin.
Pf 477736
Manufactured by Merck Group
Sourced in United States
PF-477736 is a laboratory research tool produced by Merck Group. It is a small molecule that functions as a selective inhibitor. The core function of PF-477736 is to modulate specific molecular targets in a controlled experimental setting. Detailed information on the intended applications of this product is not available within the scope of this request.
Automatically generated - may contain errors
Lab products found in correlation
Cisplatin, by Merck Group (2 mentions)
Gemcitabine, by Merck Group (2 mentions)
Azd7762, by Merck Group (2 mentions)
P chk1 317, by Cell Signaling Technology (2 mentions)
Chp134, by American Type Culture Collection (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Sk n as, by American Type Culture Collection (2 mentions)
Sk n be, by American Type Culture Collection (2 mentions)
Sms san, by American Type Culture Collection (2 mentions)
Nbl s, by American Type Culture Collection (2 mentions)
Matrigel, by Merck Group (1 mentions)
Dextrose, by Merck Group (1 mentions)
Nod scid mice, by Charles River Laboratories (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Azd7762, by Selleck Chemicals (1 mentions)
Gdc 0941, by Cayman Chemical (1 mentions)
Ly2606368, by MedChemExpress (1 mentions)
P chk1 ser296, by Cell Signaling Technology (1 mentions)
10 protocols using pf 477736
1
Xenograft Mouse Model for LIMD1 Knockout
2
Investigating CHK1 and PI3K Inhibitors
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The CHK1 inhibitors PF477736 (Sigma-Aldrich, USA), AZD7762 (Selleck Chemicals, CA, USA), and LY2606368 (MedChem Express, USA); as well as the PI3K inhibitors GDC0941 (Cayman Chemical, USA) and GSK1059615 (Cayman Chemical, USA) were used. The PI3K inhibitor BYL719 was kindly provided by Novartis Pharma AG (Basal, Switzerland). Cisplatin was purchased from Sigma-Aldrich. Antibodies against the following molecules were used for immunoblotting: CHK1, p-CHK1-Ser296, AKT, p-AKT-Ser473 (Cell Signaling Technology), and GAPDH (Bioworld, USA).
3
Combinatorial Cancer Therapy Evaluation
4
Cytotoxicity Assay with Anticancer Agents
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Camptothecin, cisplatin, etoposide, 5-fluorouracile, gemcitabine, PF477736, hydroxyurea (HU), oxaliplatin, calcein-AM, bovine serum albumin (BSA), paraformaldehyde (PFA), Triton-X100, Hoechst 33342, propidium iodide (PI), and benzonase and dimethyl sulfoxide (DMSO) were purchased from SIGMA (Saint Quentin Fallavier, France). Olaparib, VE-822, and SN38 were purchased from Selleckchem (Euromedex, Souffelweyersheim, France).
5
Evaluating Melanoma Cell Viability and Synergistic Combinations
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Cell viability was measured using the neutral red uptake assay [53 (link)]. Melanoma cells were seeded in 12-well flat bottom tissue culture plates. One-day post-seeding, the cells were treated with PLX4032 (APEXBIO), PF477736 (Sigma-Aldrich), or DMSO for 48 h. The cells were then recovered in regular media for 2–3 days. The plates were incubated for 2 h in regular medium containing 40 μg/ml of neutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride, Sigma). After the cells being washed with PBS, the dye was extracted from each well with acidified ethanol solution and the absorbance at 540 nm was read by a Multiskan Spectrum microplate spectrometer (Thermo Lab systems).
For clonogenic survival assays, cells were seeded at 5000 cells per well in 6-well plates and treated with drugs as described above. Regular media was replaced after treatment. After 10 days, cells were stained with 0.5% crystal violet in 20% methanol and counted.
Combination index (CI) for cells treated with two drugs was determined by CompuSyn software for the Chou and Talalay analysis as described [54 (link)]. A CI value less than 1 indicates synergic effect.
For clonogenic survival assays, cells were seeded at 5000 cells per well in 6-well plates and treated with drugs as described above. Regular media was replaced after treatment. After 10 days, cells were stained with 0.5% crystal violet in 20% methanol and counted.
Combination index (CI) for cells treated with two drugs was determined by CompuSyn software for the Chou and Talalay analysis as described [54 (link)]. A CI value less than 1 indicates synergic effect.
6
Neuroblastoma Cell Culture and Inhibitor Assays
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The human neuroblastoma cell lines CHP134, NBLS, SKN‐BE, SK‐N‐AS, IMR32, and SMS‐SAN were purchased from the American Type Culture Collection and the RIKEN Bioresource Cell Bank, Tohoku University Cell Resource Center.24 , 25 Cells were cultured in RPMI 1640 medium supplemented with 10% heat‐inactivated FBS, 50 µg/mL penicillin, and 50 µg/mL streptomycin (Thermo Fisher Scientific). PF‐477736 (a selective inhibitor of CHK1) and gemcitabine (GEM) or trametinib (GSK1120212, a selective inhibitor of MEK1/2) were purchased from Sigma Aldrich or Chemitek, respectively.
7
Kinase Inhibition Assay in Zygotes
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For kinase inhibitor experiments zygotes were incubated after scoring for visible pronuclei at 20 hr post-hCG (corresponding to early/mid G1 phase) in continuous presence of the respective inhibitor [10 μM KU-55933 (Hickson et al., 2004 (link)), Sigma-Aldrich; 10 μM VE-821 (Charrier et al., 2011 (link), Reaper et al., 2011 (link)), Sigma-Aldrich; 10 μM NU7026 (Veuger et al., 2003 (link)), Sigma-Aldrich; 2 mM caffeine (Sarkaria et al., 1999 (link)), Sigma-Aldrich; 100 nM AZD7762 (Zabludoff et al., 2008 (link)), Sigma-Aldrich; 5 μM NSC109555 (Jobson et al., 2007 (link)), Sigma-Aldrich; 10 nM PF-477736 (Blasina et al., 2008 (link)), Sigma-Aldrich]. Incubation in Tet3 inhibitor [1 mM Dimethyloxalylglycine (DMOG) (Amouroux et al., 2016 (link)), Sigma-Aldrich] directly followed zygote isolation at 17-18h post-hCG.
8
Cell signaling pathway analysis
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Anti-phospho-H2AX (Ser139) antibodies were purchased from Millipore (Burlington, MA, USA). Anti-cleaved PARP, anti-cleaved caspase-3, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-CHK1, anti-phospho-CHK1 (Ser345 and Ser296), anti-phospho-cdc2 (Tyr15), anti-phospho-BRCA1 (Ser1524), anti-Bcl2, anti-Bax, anti-Bak, anti-Bcl-XL, and anti-Rad51 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibodies were purchased from Sigma Aldrich (St. Louis, MO, USA). anti-Rad51 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For gene knockdown experiments, control siRNA (sc-37007), CHK1 siRNA (sc-29269), and Rad51 siRNA (sc-36361) were purchased from Santa Cruz Biotechnology. RNAiMax was purchased from Invitrogen (Carlsbad, CA, USA). PF-477736, a selective CHK1 inhibitor, and B02, a Rad51 inhibitor, were purchased from Sigma Aldrich.
9
CRC Cell Line Culturing and Compound Characterization
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Human CRC cell lines HCT-116 and HT-29 were acquired from ATCC (Manassas, VA). Both cells were grown in Dulbecco's modified Eagle's medium (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Omega Scientifics, Tarzana, CA) and 1× penicillin/streptomycin (Corning, Manassas, VA). Cell lines were tested for mycoplasma contamination before each experiment. F10 compound was synthesized according to methods described previously [21] (link). 5-FU and Chk1 inhibitors PF-477736 and prexasertib were purchased from Sellechem (Houston, TX). Both the PF-477736 and prexasertib were dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO), whereas F10 was dissolved in sterile H2O. 5-Chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU) were purchased from Sigma (St. Louis, MO) and dissolved in growth media. The following antibodies were used in this study: Santa Cruz Biotechnology (Dallas, TX): FANCD2 (catalog no. sc-20022), GAPDH (catalog no. sc-32233), Chk1 (catalog no. sc-8408), and Rad51 (catalog no. 8349); Cell Signaling (Beverly, MA): pChk1-317 (catalog no. 2344) and γH2AX (catalog no. 2577).
10
Culturing Human Neuroblastoma Cell Lines
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The human NB cell lines NB-39-nu, SMS-SAN, NBLS, CHP134, SH-SY5Y and SK-N-AS, and SK-N-BE were purchased from the American Type Culture Collection (Manassas, VA, USA) and the RIKEN Bioresource Cell Bank, Tohoku University Cell Resource Center (Miyagi, Japan). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 μg/ml penicillin, and 50 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). 293FT cell line (Thermo Fisher Scientific) was maintained in DMEM medium with 10% FBS, 50 μg/ml penicillin, 50 μg/ml streptomycin, and 500 μg/ml Geneticin (Thermo Fisher Scientific). Cells were incubated at 37°C in a 5% CO2 humidified atmosphere (MCO-175-PJ, PHC, Tokyo, Japan). PF-477736 (henceforth, CHK1i) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions were made in dimethyl sulfoxide (DMSO).
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