Cells were synchronized at G1/S phase transition by double thymidine block, as previously described.48 (link) Subsequently, at the indicated timepoints, cells were collected for FACS and western blot analysis. Briefly, synchronized cells were centrifuged and ressuspended in ice-cold PBS and fixed under gentle vortexing by dropwise addition of an equal volume of ice-cold 80% ethanol (−20 °C), followed by 30 min on ice. Subsequently, samples were stored at 4 °C for at least 18 h, followed by incubation with RNase A solution (10 μg/ml, in PBS) for 30 min at 37 °C. Finally, propidium iodide (25 μg/ml) was added to the samples for 5 min, before flow cytometry analysis. In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF-κB inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μM, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.
Xmd8 92
Manufactured by Bio-Techne
Sourced in United Kingdom
XMD8-92 is a small molecule inhibitor that targets the X-linked inhibitor of apoptosis (XIAP) protein. It is a laboratory research reagent intended for use in experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Bix02189, by Bio-Techne (2 mentions)
Bay11 7085, by Santa Cruz Biotechnology (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Xmd17 109, by MedChemExpress (1 mentions)
Docetaxel, by Selleck Chemicals (1 mentions)
Doxorubicin, by Selleck Chemicals (1 mentions)
Auy922, by Selleck Chemicals (1 mentions)
Matrigel, by BD (1 mentions)
Sapk jnk, by Cell Signaling Technology (1 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Phospho sapk jnk thr183 tyr185 81e11, by Cell Signaling Technology (1 mentions)
Phospho p90rsk thr359 ser363 antibody, by Cell Signaling Technology (1 mentions)
Rsk1 d6d5, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204 d13.14.4e, by Cell Signaling Technology (1 mentions)
On targetplus non targeting control pool sirna, by Horizon Discovery (1 mentions)
Alexa fluor conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Anti erk2 antibody, by Santa Cruz Biotechnology (1 mentions)
Bix02189, by Selleck Chemicals (1 mentions)
8 protocols using xmd8 92
1
Cell Cycle Synchronization and Analysis
2
Inhibiting Autophagy and MEK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise stated, cells were treated with 10 µM BIX02189 (Tocris 4842) and 10 µM XMD8–92 (Tocris 4132) for 16 h and with 50 nM Bafilomycin A1 (Sigma B1793) for 2 h.
3
Synergistic Anticancer Effects of 5-FU and XMD Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5-FU (Sigma-Aldrich), XMD8-92 (Tocris Bioscience, Bristol, UK) and XMD17-109 (MedChem Express, NJ, USA) stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at −80°C. Before treatments, cells were allowed to adhere for 24 h, and then incubated with either 5-FU alone or in combination with XMD8-92 or XMD17-109, at the indicated concentrations, for the indicated times. For cytotoxicity assays, HCT116 cells were seeded in 96–well plates, at 1 × 104 cells/well, and next exposed to 8–200 μM 5-FU and/or 4 μM XMD8-92 or 2 μM XMD17-109 for 48 h. For the morphological detection of apoptotic nuclei, stably transduced HCT116 cells were seeded into 35 mm dishes, at a density of 1.5 × 105 cells/dish, and exposed to 8–200 μM 5-FU for 24 h. For the Guava Nexin assay, HCT116 cells were seeded in 24-well plates, at 5 × 104 cells/well, and exposed to 8–50 μM 5-FU and/or 4 μM XMD8-92 or 2 μM XMD17-109 for 48 h. For the evaluation of caspase-3/7 activity, HCT116 cells were seeded on a 96-well plate at 1.5 × 105 cells/well, and exposed to 8–200 μM 5-FU and/or 4 μM XMD8–92 or 2 μM XMD17-109 for 16 h. All experiments were performed in parallel with DMSO vehicle control. Final DMSO concentration was always 0.1%.
4
In Vivo TNBC Tumor Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The local animal ethics committee gave approval for use of the mice. Female balb/c nude mice at 5 weeks of age (Animal Resources Centre, WA, Australia) were inoculated in the mammary fat-pads with exponentially growing MDA-MB-231 TNBC cells (5×106 per fat pad) in 50 µL of 50:50 PBS:Matrigel™ (BD Biosciences, CA, USA). Treatments started when tumors were 50 ± 1 mm3 as calculated from caliper measurement of tumor's longest (a) and shortest (b) diameters: tumor volume (mm3) = a/2 × b2. Docetaxel, doxorubicin and the Hsp90 inhibitor AUY922 were purchased from Selleck Chemicals and the Erk5 inhibitor XMD 8-92 from Tocris Biosciences. All drugs were diluted in 5% solution of D-glucose in PBS for intraperitoneal injection. Doses and schedule of treatments are detailed in Figure legends. Mice were monitored for change in weight and other toxicity indications and none were observed. Tumor growth was monitored by caliper measurements to calculate the change in tumor volume compared to day 0. Six tumors were excised on day 13 to perform ex vivo studies as below.
5
Autophagy Modulation by ULK1/2 Inhibitors
6
Antibody Characterization for Signaling Pathways
7
Macrophage Inhibitors Pretreatment Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In some experiments, macrophages were pretreated with the following inhibitors: BIX 02189 (Tocris), a selective inhibitor of MEK5; XMD 8-92 (Tocris), an inhibitor of ERK5; and 10058-F4 (Tocris), a c-Myc-Max dimerization inhibitor.
Optimal doses of inhibitors were determined for each experiment. Mock pretreatment was performed with vehicle alone (DMSO) at a maximum concentration of 0.05%.
Optimal doses of inhibitors were determined for each experiment. Mock pretreatment was performed with vehicle alone (DMSO) at a maximum concentration of 0.05%.
8
Kinase Inhibition Assay in EGF-Stimulated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in a 6-well plate (250,000 cells/well) for 24 hrs. To examine kinase activity or inhibition, the cells were serum-starved for 18-24 h. The inhibitors XMD8-92 (Tocris, Minneapolis, MN) and trametinib (Selleckchem, Houston, TX) were added for 30 minutes prior to EGF (100ng/ml) stimulation for 15 minutes as previously described [21 ]. Cells were lysed and examined for kinase activation using standard western blot procedures.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!