Ruxolitinib

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Ruxolitinib is a small molecule inhibitor that selectively targets the Janus kinase (JAK) enzymes, particularly JAK1 and JAK2. It is commonly used for research purposes in cell and animal studies to investigate the role of JAK signaling in various biological processes.

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Lab products found in correlation

Tnt quick coupled transcription transcription system, by Promega (1 mentions) Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions) Methylcellulose, by Merck Group (1 mentions)

3 protocols using ruxolitinib

Phosphorylation and Dephosphorylation of STAT5

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For the phosphorylation assay, STAT5 (produced in reticulocyte lysate) was incubated for 15 min at 30 °C in the presence or absence of HSP27 (produced in reticulocyte lysate) with JAK2 recombinant kinase (50 ng, 14-640, Merck Millipore) in a 15 µl of assay kinase buffer (Hepes pH 7.4, 10 mM, NaCl 50 mM, MgCl₂ 5 mM, MnCl₂ 5 mM, DTT 1 mM, NaF 10 mM) in the presence or absence of ATP (250 µM, #9804, Cell Signalling Technology) and the reaction was stopped by adding 2× Laemmli buffer.
In vitro transcription and translation reactions were performed using TNT Quick Coupled Transcription/Transcription System (L1170, Promega) as recommended by the supplier. Next, 1 µg of the plasmid DNA template was transcribed and the protein was translated at 30°C for 90 min.
For the de-phosphorylation assay, a phosphorylation kinase assay JAK2/STAT5 was first done in the presence of ATP as described above and the reaction was stopped by adding 20 µM of Ruxolitinib (sc-396768A, Santa-Cruz) for 5 min. Then, HSP27 (produced in reticulocyte lysate) was added or not to the samples and incubated for 10 min at 30 °C. The recombinant SHP2 (590 ng, SRP0217, Sigma-Aldrich) was added or not to the samples for 12 min at 30 °C and the reaction was stopped by adding 2× Laemmli buffer.

Sevin M., Kubovcakova L., Pernet N., Causse S., Vitte F., Villeval J.L., Lacout C., Cordonnier M., Rodrigues-Lima F., Chanteloup G., Mosca M., Chrétien M.L., Bastie J.N., Audia S., Sagot P., Ramla S., Martin L., Gleave M., Mezger V., Skoda R., Plo I., Garrido C., Girodon F, & de Thonel A. (2018). HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis. Nature Communications, 9, 1431.

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Inhibition of JAK1 Signaling in COV434 Cells

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The commercially available inhibitor Ruxolitinib (CAS 941678-49-5, Santa Cruz, Dallas, USA) was used for inhibition of JAK1 signalling in COV434 cells. The manufacturer’s mechanism of action for Ruxolitinib, involves competitive binding to the JAK1 receptor, disabling JAK phosphorylation and preventing downstream signalling to STAT proteins. The appropriate inhibitor concentrations and treatment length were based on the IC50 of Ruxolitinib and were optimised specifically for COV434 cells (data shown in Supplementary Figure 4). Cells were treated for 72 hr as COV434 cells are slow-growing and require time to cell cycle for proliferation effects to be examined (Pastuschek et al. 2015 (link)). This treatment length also demonstrated the most robust decrease in JAK1 phosphorylation, compared with 24 hr and 48 hr treatments (data not shown). COV434 cells to be treated with Ruxolitinib for collection were plated at a density of 400,000 cells/well in 6-well plates and grown overnight. The next day, 4 mL of media containing 100 nM Ruxolitinib was added to the cells, and cells were treated for 72 hr. Following treatment, the cells were counted and were either centrifuged into cell pellets for RNA or protein extraction or fixed using paraformaldehyde and spotted onto glass slides for immunocytochemistry (ICC).

Frost E., Ford E., Peters A., Reed N., McLaughlin E., Baker M., Lovell-Badge R, & Sutherland J. (2020). STAT1 and STAT3 are expressed in the human ovary and have JAK1-independent functions in the COV434 human granulosa cell line. Reproduction, fertility, and development, 32(12), 1027-1039.

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Ruxolitinib Cell Culture Protocol

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Ruxolitinib (CAS 941678-49-5) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Methyl cellulose, (product number : M7027) for the tumor sphere assay and radioimmunoprecipitation assay (RIPA) Lysis Buffer System (sc24948) were obtained from Sigma Aldrich (St. Louis, MO, USA) and Santa Cruz Biotechnology, respectively. The PureLink^® RNA Mini Kit (Life Technologies, Grand Island, NY, USA), High-Capacity cDNA Reverse Transcription Kit (Life Technologies), TaqMan™ Universal PCR Master Mix (Life Technologies), and molecular grade water were obtained from Thermo Fisher Scientific (Rochester, NY, USA).

Delen E, & Doğanlar O. (2020). The Dose Dependent Effects of Ruxolitinib on the Invasion and Tumorigenesis in Gliomas Cells via Inhibition of Interferon Gamma-Depended JAK/STAT Signaling Pathway. Journal of Korean Neurosurgical Society, 63(4), 444-454.

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