Hi everyone,
I plated HeLa cells at 2 × 10⁵ cells/mL and treated them with or without GSK963 (100 nM).
- For apoptosis induction, I used 50 or 100 ng/mL TNFα + 1 μM birinapant.
- For necroptosis induction, I used 50 or 100 ng/mL TNFα + 1 μM birinapant + 20 μM zVAD.fmk.
However, the Western blot results did not show any inhibition of MLKL or RIPK3 expression. Has anyone experienced a similar issue or could suggest why GSK963 might not work under these conditions?
Thanks in advance for any insights!
Hi Fan Janice
GSK963 can inhibit TNF-α–induced necroptosis, but note that apoptosis and necroptosis are distinct processes. To accurately distinguish between them, it is recommended to monitor specific markers such as RIPK1, RIPK3, and MLKL. At present, I have not found literature showing that treatment with GSK963 after TNF-α–induced necroptosis affects levels of Cleaved-Caspase-3 or Cleaved-PARP.
You may, however, refer to studies where GSK963 was tested in other cell lines, showing reduced protein levels of p-RIPK1, RIPK3, and MLKL (e.g., PMID: 27551444). It would be useful to include these markers in your own follow-up experiments.
Additionally, when preparing your solutions, please note that the product is poorly soluble in water. For diluting from 100 mM to 100 μM, use DMSO to avoid precipitation. From 100 μM to 100 nM, you can dilute 1,000-fold directly with culture medium. If you are unsure whether the final working solution is fully dissolved, check under a microscope for irregular crystals. If precipitation occurs, you may use an ultrasonic bath at 37 °C to help dissolve it.
The answer to this question is provided by MedChemExpress Technical Support.
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