The Library of Pharmacologically-Active Compounds (LOPAC), 6α-methylprednisolone (6MP), dexamethasone (Dex), mifepristone, PD166285 (PD16), PD173952 (PD17), NU6027 and CGP7454A were purchased from Sigma-Aldrich; dasatinib, MK-1775 and CHIR-124 from SelleckChem. All compounds were diluted in DMSO to 10 mM stock solutions and kept at -20°C.
Chir 124
Manufactured by Selleck Chemicals
Sourced in United States
CHIR-124 is a lab equipment product manufactured by Selleck Chemicals. It functions as a small molecule inhibitor.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Mk 1775, by Selleck Chemicals (3 mentions)
Ve 821, by Selleck Chemicals (3 mentions)
Bi2536, by Selleck Chemicals (2 mentions)
Hydroxyurea, by Merck Group (2 mentions)
Thymidine, by Merck Group (2 mentions)
Ku55933, by Selleck Chemicals (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Pf 477736, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dasatinib, by Selleck Chemicals (1 mentions)
Mifepristone, by Merck Group (1 mentions)
Nu6027, by Merck Group (1 mentions)
Anti cdc25c c 20, by Santa Cruz Biotechnology (1 mentions)
Anti cdc25a, by Cell Signaling Technology (1 mentions)
Anti cdc25b, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Guanosine, by Merck Group (1 mentions)
Cytosine, by Merck Group (1 mentions)
14 protocols using chir 124
1
Pharmacologically-Active Compound Library
2
Establishing Docetaxel-Resistant Cell Line
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The IGR-CaP1 cell line was maintained in RPMI1640 medium supplemented with 10% FBS. Docetaxel-resistant clones were selected by exposing cells to Docetaxel in a dose-escalation manner as described [33 (link)]. Surviving clones to low dose of Docetaxel were subsequently subjected to 5nM, 12nM, 25nM, 50nM, 100nM and 200nM of Docetaxel. Cells freely dividing in each dose of Docetaxel-containing media were considered resistant. Docetaxel (TAXOTERE®) was kindly provided by Sanofi-Aventis (France). NSC663284 was purchased from Calbiochem; BI2536 and CHIR-124 were purchased from Selleckchem and were resuspended in DMSO. Anti-LZTS1 (C-20), and anti-CDC25C (C-20) were obtained from Santa-Cruz Biotechnology, anti-CDC25A, anti-CDC25B, from Cell Signalling, anti-GAPDH and anti-β-actin from Sigma.
3
Nucleoside and Inhibitor Resuspension Protocol
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Hydroxyurea (Sigma; #H8627) was resuspended directly in R15 media at 3 mM . Adenosine (Sigma; #A9251), cytosine (Sigma; #C3506), thymidine (Sigma; #T9250) and uridine (Sigma; #U3750) were resuspended at 3 mM in UltraPure distilled water (Invitrogen, Carlsbad, CA, USA; #10977-015). Guanosine (Sigma; #G6752) was resuspended at 30 mM in DMSO. Both VE-821 and CHIR-124 (Selleckchem, Boston, MA, USA; #S8007 and #S2683, respectively) were resuspended in DMSO at 10 mM .
4
Inhibition of CHEK1 in Cell Lines
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The following reagents and primary antibodies are used in this study: DMEM-F12 (Gibco, 10565-018), fetal bovine serum (Gibco, 10082-147), Accutase solution (Sigma, A6964-100), Alamar Blue (Invitrogen, DAL1100), RIPA buffer (Sigma, R0278), phosphatase inhibitor cocktail (Sigma, P0044), protease inhibitor cocktail (P8340), Bradford (BIORAD, 500-0006), BSA used in Bradford assay (BioLabs, B9001S), PageRuler plus prestained protein (Thermo scientific, 26619), iScript Reverse Transcription supermix for qRT-PCR (Bio-rad, 170-8841), shCHEK1 lentivirus particles (pGFP-C-shLenti, Origene, TR320302), CHIR-124 (Selleckchem, S2683), anti-CHEK1 (Novus Biologicals, NB100-464, rabbit, used for WB and IHC), anti-RAD54L (Novus Biologicals, NBP2-33916, rabbit, used for WB), and β-Actin (Sigma, A5316, mouse, used for WB).
5
Temporal control of cell fate in zebrafish
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For Mtz treatment, the Tg(lfabp:DenNTR) transgenic larvae at 5 dpf were incubated with 10 mM Mtz (metronidazole, Sigma) for 24 hours. For ethanol (EtOH) treatment, larvae were treated with 1.5% EtOH (Sigma Aldrich) for 48 hours, starting from 4 dpf17 (link). For HU (hydroxyurea, Sigma) treatment, embryos were treated with 100 mM HU in egg water from R0h to R8h. Larvae were treated with 0.1 μM Chk1i (CHIR-124, Selleck), 5 μM ATRi (VE-821, Selleck), 2 μM ATMi (KU-55933, Selleck) and 20 μM Mirin (Selleck) from R8h to R48h. The chemical solutions were renewed every 24 h to maintain the pharmacological effects. And a 0.2% DMSO solution in egg water was used as a control. When chemical treatment is removed, the larvae need to be washed and recovered in the egg water.
For temporal control of CreER activities, larvae were collected and incubated with 5 μM 4-hydroxytamoxifen (4-OHT, Sigma) for 24 h from 4 to 5 dpf, followed by three washes with egg water.
The larvae of Tg(hsp70l:nbn-p2A-mCherry)cq141 were heat-shocked at 38.5 °C for 40 min at the indicated time frame and returned to 28.5 °C for further analysis.
For BODIPY feeding assay, larvae were fed with BODIPY FL C5 (Invitrogen) for 4 h at 5 dpf and then live imaging, as previously described26 (link).
For temporal control of CreER activities, larvae were collected and incubated with 5 μM 4-hydroxytamoxifen (4-OHT, Sigma) for 24 h from 4 to 5 dpf, followed by three washes with egg water.
The larvae of Tg(hsp70l:nbn-p2A-mCherry)cq141 were heat-shocked at 38.5 °C for 40 min at the indicated time frame and returned to 28.5 °C for further analysis.
For BODIPY feeding assay, larvae were fed with BODIPY FL C5 (Invitrogen) for 4 h at 5 dpf and then live imaging, as previously described26 (link).
6
Cell Culture Conditions for Hematological and Embryonic Cell Lines
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Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2, except for mouse pre-B cells (7.5% CO2). Human Burkitt lymphoma, Nalm6 cells and murine pre-B cell lines were cultured in RPMI-1640 medium (Sigma-Aldrich, R0883) supplemented with 10% FCS (Gibco, 10270-106), 2 mM l -glutamine (Sigma, G7513), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, P0781) and 50 μM 2-mercaptoethanol (Sigma, M3148). For murine pre-B cells and primary bone marrow culture, 1 mM sodium pyruvate (Gibco, 11360-039), 1× non-essential amino acids (Gibco, 11140-35) and IL-7 (supernatant of IL-7 expressing J558L cells) was added in addition. Primary FACS-sorted murine pro B cells were cultured in DMEM medium (Sigma, D5671), supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM l ‐glutamine, 10 mM Hepes (LONZA, BE17-737E), 1 mM sodium pyruvate, 1× non-essential amino acids and 50 μM 2-mercaptoethanol. Human embryonic kidney cells (HEK293T) were grown in DMEM medium, supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin and 2 mM l ‐glutamine. Reagents: PF-477736 (Selleckchem S2904), CHIR-124 (Selleckchem S2683), QVD (SML0063, Sigma), DMSO (D5879, Sigma), Doxycycline (D9891, Sigma), anti-mouse CD40 (102908, biolegend, HM40-3).
7
Modeling SLE B Cell Responses
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B cells from healthy individuals were cultured following isolation from fresh PBMCs (EasySep Human B Cell Isolation Kit) in a 37°C humidified incubator with 5% CO2 while plated in a 96-well round-bottom plate (Sarstedt) in a concentration of 1.5 × 105 cells per well. Cells were cultured in RPMI 1640 with GlutaMAX (catalog no. 61870036, Gibco) supplemented with 10% (v/v) heat-inactivated FBS (catalog no. 10270106, Gibco), penicillin-streptomycin (100 U/ml and 100 μg/ml, respectively; catalog no. 15140m, Gibco), sodium pyruvate (1 mM; catalog no. 11360070, Gibco), Hepes (10 mM; catalog no.15630106, Gibco), and 2-mercaptoethanol (0.05 mM; catalog no. 31350010, Gibco). In addition, all cultured B cells, irrespective of experiment, were supplemented with a “survival/mild proliferation” cocktail of IL-21 (50 ng/ml; catalog no. 200-21, PeproTech), CpG-B (2.5 μg/ml; catalog no. HC4039, Hycult Biotech), and sCD40L/CD154 (1 μg/ml; catalog no. 11343345, ImmunoTools). To mimic DDR in SLE B cells, IFN-α (catalog no. 11200-1, PBL Assay Science) was added to cells at 850 U/ml. For chemical ATR inhibition, berzosertib (i.e., ATRi; catalog no. S7102, Selleckchem) was added to cells at 2 or 5 μΜ, and for Chk1 inhibition, CHIR-124 (i.e., Chk1i; catalog no. HY-13263, Selleckchem) was added to cells at 50 nM (details on legends).
8
Characterization of Pancreatic Cancer Cell Lines
9
Clonogenic Survival Assays for Drug Efficacy
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Clonogenic survival assays were performed as previously described (10 (link)). The day prior to treatment, 300–500 cells were seeded in complete medium in 60 mm plates. The day following seeding, cells were treated for varying times depending on the drug in serum-free medium. Cisplatin (Sigma-Aldrich; 479306) was prepared daily as a fresh 1 mmol/l stock in PBS. All Cisplatin treatments in clonogenic assays were performed for two hours. M6620 (Selleckchem; S7102), VX-803 (Selleckchem; S9639), MK-1775 (Selleckchem; S1525), BMN 673 (Selleckchem; S7048), KU-55933 (Selleckchem; S1092) and CHIR-124 (Selleckchem; S2683) were prepared in DMSO, and treatments were performed for 4 h. Mitomycin C (Selleckchem; S8146) was prepared in DMSO and treatments were performed for 2 h. NERx329 (compound 43) was synthesized as described (17 (link)). Once colonies reached a size of at least 50 or more cells, plates were washed once with PBS, and crystal violet was added (20% ethanol, 1% w/v crystal violet). After counting, colony assay data were plotted and IC50s estimated using Sigma Plot version 10.0 or GraphPad Prism. For synergy studies, a constant Cisplatin:M6620 or Cisplatin:NERx329 ratio was used based upon the approximate IC50 value for each drug in H1299 ERCC1Δ cells. CI values for synergy studies were generated in CompuSyn and data plotted in GraphPad Prism (GraphPad Software, CA).
10
Antibodies and Inhibitors for Cell Signaling
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Primary Antibodies against CHK1 (2G1D5), PARP1 (46D11), H2AX (D17A3), and ɣH2AX (20E3) were purchased from Cell Signaling; against GAPDH (0411), HA (12CA5) and Myc (9E10) were purchased from Santa Cruz Biotechnology. Anti-STAU2 (HPA019155), anti-β-Actin (A5441), anti-Flag (F3165), and HRP-Streptavidin were obtained from Sigma. All primary antibodies were used at 1:1000 dilution. MG132 (C2211), iCHK1 (681637) and DMSO were purchased from Millipore-Sigma; ZVAD-FMK (S7023), Emricasan (S7775), PF47 (PF-477736, S2904), and CHIR124 (S2683) were obtained from Selleckchem.
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