For assessment of resistance of CRC cells to chemotherapy, CRC cells were treated with oxaliplatin (0, 0.625, 1.25, 2.5, 5, 10 and 20 μM) (Sigma-Aldrich, USA) for 72 h. The cell viabilities then were detected by CCK8 assay and the half-maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism 5 software. For the drug-induced cytotoxicity analysis, CRC cells were treated with 5 μM oxaliplatin for 72 h. Subsequently, the cells were harvested and performed apoptosis detection by a FACS Aria cytometer (BD Bioscience) using the Annexin V-PE and 7-amino-actinomycin D (7-AAD) double-staining Apoptosis Detection kit (KeyGEN). For Tws119 (Selleck, USA) usage, CRC cells with 2 μM Tws119 treatment for 72 h were harvested for western blot analysis.
Tws119
Manufactured by Selleck Chemicals
Sourced in United States
TWS119 is a laboratory equipment product designed for scientific research and analysis. It serves as a tool for performing various tasks in a controlled environment. The core function of TWS119 is to provide a stable and precise platform for conducting experiments and measurements.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
Fabp4, by Cell Signaling Technology (1 mentions)
Pparγ, by Cell Signaling Technology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
Gapdh, by Proteintech (1 mentions)
Galunisertib ly2157299, by Selleck Chemicals (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Wortmannin, by Beyotime (1 mentions)
N acetylcysteine, by Beyotime (1 mentions)
Ly294002, by Promega (1 mentions)
Cell culture materials, by Corning (1 mentions)
Gsk3β kinase activity quantitative detection kit, by Genmed Scientifics (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Oxaliplatin, by BD (1 mentions)
Oxaliplatin, by Merck Group (1 mentions)
16 protocols using tws119
1
Assessing Chemotherapy Resistance in CRC
2
Effects of AGEs and β-phosphoglycerine on HASMCs
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HASMCs were obtained from ScienCell American and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL) in a 37 • C incubator with humidified air containing 5% CO 2 . The HASMCs were previously characterized [16] . The culture medium was replenished twice per week. First, HASMCs were divided into the following three groups: HASMCs were treated with vehicle (control group), AGEs (Biovision, Japan), β-phosphoglycerine (10 mM) (Beyotime Biotech-nology, Shanghai, China), or AGEs + β-phosphoglycerine for 5 days. Also, the following five groups were designed:
(1) the blank control group, (2) the dimethyl sulfoxide (DMSO, vehicle) group, (3) the AGEs group, in which cells were treated with 25 µg/mL AGEs in culture for 5 days, (4) the LY294002 group, in which cells were pretreated with LY294002 (Selleck, USA), an AKT inhibitor, at 20 µM for 2 hours followed by AGEs treatment, and (5) the TWS119 group, in which cells were pretreated with TWS119 (Selleck, USA), an inhibitor of GSK3-β, at 10 µM for 2 hours followed by AGEs treatment. HASMCs of passages 3-6 were used for this study.
(1) the blank control group, (2) the dimethyl sulfoxide (DMSO, vehicle) group, (3) the AGEs group, in which cells were treated with 25 µg/mL AGEs in culture for 5 days, (4) the LY294002 group, in which cells were pretreated with LY294002 (Selleck, USA), an AKT inhibitor, at 20 µM for 2 hours followed by AGEs treatment, and (5) the TWS119 group, in which cells were pretreated with TWS119 (Selleck, USA), an inhibitor of GSK3-β, at 10 µM for 2 hours followed by AGEs treatment. HASMCs of passages 3-6 were used for this study.
3
Adipocyte Differentiation Protein Analysis
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TRIzol reagent was purchased from Invitrogen. Fast-start universal SYBR Green master mix was from Roche. The inhibitor TWS119 was from Selleck. The antibodies used for immunoblotting (IB): PPARγ (Cell Signaling, #2443), FABP4 (Cell Signaling, #3544), TauT (Absin, abs140562a), β-catenin (Cell Signaling, #8480), β-actin (Santa Cruz, sc-47778), GAPDH (Proteintech, 60004-1-Ig), α-Tubulin (Santa Cruz, sc-58667).
4
Inhibitors for Cancer Cell Signaling
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PcTx1 (a potent and selective ASIC1α blocker, Psalmotoxin 1) and GN25 (a novel inhibitor of Snail‐p53 binding) were procured from ApexBio Technology (Houston, USA). MK2206 (a specific inhibitor of AKT), TWS119 (a specific inhibitor of GSK3β) and Galunisertib (LY2157299) (TGF‐β/Smad inhibitor) were obtained from Selleck Chemicals (Houston, TX, USA). C19 (an inhibitor of EMT) was obtained from Shanghai Dongcang Biological Technology Co., Ltd. (Shanghai, China). OXA and 5FU were obtained from Sigma‐Aldrich (St. Louis, USA). These reagents and chemicals were dissolved in dimethyl sulfoxide at −80℃ until use and then diluted to a suitable working concentration with RPMI‐1640 medium. TGF‐β1 was obtained from PeproTech (Rocky Hill, NJ). TRIzol reagent was bought from Invitrogen Corp. (Carlsbad, CA, USA). First Strand cDNA Synthesis Kit was acquired from Thermo Fisher Scientific (Waltham, MA, USA).
5
ROS Regulation in Cellular Assays
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Cell culture materials were obtained from Corning (Shanghai, China). A ROS assay kit, Wortmannin, N-acetyl cysteine (NAC), and H2O2 were purchased from Beyotime Biotechnology (Shanghai, China). TWS119 was purchased from Selleck (Texas, U.S.A.). LY294002 was purchased from Promega (Madison, U.S.A.). Salubrinal was obtained from MCE China. CD107a (LAMP-1) ELISA kit were purchased from ElabScience (Wuhan, China), whereas, PGE2 and IFN-γ ELISA kits was purchased from Mlbio (Shanghai, China), and mouse RPS19 ELISA kits were purchased from SaiDongnan (Tianjin, China). Antibodies were purchased as follows: NKG2D and ULBP1 or Mult-1 (Proteintech, America), GSK-3β (Boster, Wuhan, China), pSer9-GSK-3β (Santa Cruz, CA, USA), Rae1 (Santa Cruz), H60 (R&D system, Minnesota, USA), pSer535-eIF2B (Eno Gene, Nanjing, China), and β-actin (Santa Cruz).
6
Intravitreal Injections for Tau and GSK3β Modulation
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Intravitreal injections were performed as previously described [15 (link), 25 (link)]. Briefly, mice were anesthetized by intraperitoneal injections of 2 g/kg urethane. Pupils were dilated with 1% atropine sulfate. Intravitreal injections were performed using a Hamilton syringe fitted with a 30-gauge glass microneedle under a dissecting microscope. One microliter of the solution was slowly injected into the vitreous chamber of the eye. For selected tau or GSK3β depletion, si-Tau or si-GSK3β (2 μg/μl) was injected intravitreally into the right eye and si-sc was injected into the left eye. For GSK3β inhibition, a GSK3β specific inhibitor TWS119 (Selleck, Trenton, NJ, USA; 10 nM final concentration), or vehicle (0.5% DMSO) was dissolved in 0.9% NaCl and injected intravitreally into the right eye or left eye, respectively.
7
Inhibition of LvGSK3β Protects Shrimp from WSSV
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LvGSK3β was inhibited both on the enzyme activity and mRNA expression levels by injecting with the specific inhibitor and dsRNA. Specific inhibitor of LvGSK3β, named TWS-119 (Selleck Chemicals, USA) was used with the dose refer to the Inhibitory concentration 50 (IC50) (33 (link)). At 4, 8 and 12 hpi, the hemocytes from each group were sampled for the detection of inhibition efficiency of LvGSK3β activities, using the GSK3β Kinase Activity Quantitative Detection Kit (Genmed, China). The expression of LvGSK3β was inhibited by RNAi as described above in Section RNA Interference. For the mortality test, L. vannamei were injected with 50 µl PBS containing or not containing 106 WSSV after 8 h of TWS-119 injection or 48 h of dsRNA injection. Cumulative mortality was recorded every 4 h and the differences between groups were analysed using the Mantel–Cox (log-rank χ2 test) method with the software GraphPad Prism. To investigate the genome copies of WSSV, total DNA was extracted from muscle at 48, 72 and 96 hpi and absolute real-time PCR was performed using primers WSSV32678-F/WSSV32753-R and a Taq Man fluorogenic probe, as in a previous study (34 (link)).
8
Preparation and Characterization of Ti Nanoparticles
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Ti nanoparticles were purchased from Nanjing Emperor Nano Materials Company. The nanoparticles were first stored at 300 °C for 12 h. Then, it was soaked in 75% ethanol for 2 days to remove the residuary endotoxins. Ti nanoparticles characteristics were assessed through a scanning electron microscope (SEM). Frequency distribution of diameters was analyzed by ImageJ. TWS119 (Selleck Chemicals, Houston, USA) and GANT58 (APExBIO Technology LLC, Houston, USA) were dissolved in dimethyl sulfoxide (DMSO, Sigma) at a concentration of 100 mM and 40 mM, respectively, and stored at − 20 °C.
9
Solubilizing ISX-9 for in vitro and in vivo studies
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ISX-9 (MCE, HY-12323) was solubilized by DMSO in vitro experiments and by adding 10% DMSO followed by 90% (20% SBE-β-CD in saline) in vivo experiments. ERK inhibitor: U0126-EtOH (IU0160, Solarbio). GSK-3β inhibitor: TWS 119 (Selleck, S1590). MSAB (MCE, HY-120697) is a β-catenin inhibitor.
10
Generation of T Stem Cell Memory Cells
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The CD44lowCD62Lhigh cells were stimulated with 2 μg/mL anti-CD3 (BD Pharmingen), 1 μg/mL anti-CD28 (BD Pharmingen), and 10 ng/mL IL-2 (Peprotech, Rocky Hill, NJ) in the presence of TWS119 (7 μM) (Selleckchem, Houston, TX) or Wnt3A protein (1 μg/mL) (Peprotech) in vitro. For generation of TSCMs in vivo, 2×106 OT-I naive CD8+ T cells were adoptively transferred into congenic CD45.1 mice and then injected intraperitoneally (500 μg) per mouse OVA (Sigma-Aldrich) with complete Freund’s adjuvant (CFA) (Sigma). Mice received 4 doses per day of TWS119 at 40 mg/kg from day 0 to day 3. Six days after injection, mice with or without the treatment of TWS119 were sacrificed for further analysis. The CD8+ TSCMs were isolated by flow cytometry on the basis of the expression of surface markers (CD3+ CD4− CD8+ CD62L+ CD44− CD122+ Sca-1+ T cells for in vitro-generated TSCMs or CD45.1+ CD8+ CD62L+ CD44−CD122+ Sca-1+ T cells for in vivo-generated TSCMs).
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