A Sensitive IHC Method for Monitoring Autophagy-Specific Markers in Human Tumor Xenografts

Figure 2

Sections of HCT116 xenograft tumor from nude mice treated with vehicle or investigational mTOR inhibitor MLN0128 were stained with rabbit monoclonal anti-pS6 and anti-LC3b antibodies. (a) Standard IHC staining of pS6 in vehicle-treated xenograft samples showed strong pS6 signal, whereas standard IHC staining of pS6 in MLN0128 treated xenograft (5 mg/kg for 1 hour) samples showed reduced pS6 levels compared to vehicle-treated samples. Amp HQ staining of LC3B in sections of vehicle-treated xenograft samples showed low levels of LC3B puncta staining predominately in the tumor cell cytoplasm, whereas Amp HQ staining of LC3B in sections of MLN0128-treated xenograft (5 mg/kg for 1 hour) samples showed greatly increased levels of LC3B puncta staining compared to vehicle-treated sections, predominately in the tumor cell cytoplasm. (b) Time course of pS6 response following vehicle or MLN0128 treatment (5 mg/kg) of nude mice bearing HCT116 xenograft tumors. Images were analyzed by positive pixel analysis. The data indicated that pS6 was significantly reduced at all time points (). (c) Time course of LC3B response following vehicle or MLN0128 treatment (5 mg/kg) of nude mice bearing HCT116 xenograft tumors. Positive pixel image analysis showed increased LC3B expression within 30 minutes with Amp HQ staining (). (d) Western blot analysis indicating increasing levels of lipidated LC3B (LC3B-II) as early as 0.5 hours after exposure to MLN0128.

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