Autophagy Inhibitor, 3-MA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
It is an autophagosome inhibitor.	Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line

Upstream tips
It is an autophagosome inhibitor.
Protocol tips
Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line

Publication protocol

3-Methyladenine (3MA) was purchased from Sigma-Aldrich (St. Louis, USA) and dissolved in phosphate-buffered saline (PBS, Gibco BRL) at a concentration of 100 mM. Before use, stock solutions were diluted to the required concentrations in culture medium.

Tumorigenicity in nude mice
BALB/C nude mice (4–5 week-old) were provided by the animal center at the Cancer Institute at the Model Animal Research Center of Nanjing University (Nanjing, China) and randomly divided into four groups (control group, Ib group, 3MA group, and Ib + 3MA group). U87 cells (2 × 106) in 100 μL of serum-free DMEM were injected into the right flank of mice. Tumor volume was assessed every 3 days. Mice were injected intraperitoneally (i.p.) every other day, starting on day 3, with PBS alone (control), ibrutinib (6 mg/kg/d), 3MA (30 mg/kg/d), or ibrutinib (6 mg/kg/d) and 3MA (30 mg/kg/d). The tumor and body weights were determined. Tumor volume was calculated by the following formula: (short diameter)2 × (long diameter)/2. Mice were humanely sacrificed on day 22. For immunohistochemical analysis, samples from each group of mice were stained with H&E and a primary antibody (rabbit anti-LC3A/B or rabbit anti-Ki67, both at 1:200 dilution).

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Manufacturer protocol

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