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

Figure 4

HCT116 doxycycline-inducible ATG7shRNA xenograft tumors were collected from tumor-bearing nude mice after 3, 7, and 14 days with or without doxycycline (DOX) treatment. One section from each tumor was stained for ATG7 and p62 by standard IHC methods and LC3B and NBR1 by Amp HQ IHC methods. Quantitative whole image analysis was performed using positive pixel count per total viable tumor area. (a) IHC images of ATG7, LC3B, p62, and NBR1 stained sections from xenograft tumors showed a decrease signal in ATG7 and LC3B accompanied by an increase of p62 and NBR1 signals after 14 days of DOX treatment compared with the control group (without DOX treatment). (b) Positive pixel analysis of sections stained for ATG7 by standard IHC showed a statistically significant decrease in ATG7 staining () after 7 days of DOX treatment. (c) Positive pixel analysis of sections stained for LC3B with Amp HQ IHC showed a statistically significantly decrease in LC3B puncta staining after 7 and 14 days of DOX treatment (). (d) Positive pixel analysis of sections stained for p62 by standard IHC showed an increase in p62 expression after 7 and 14 days of DOX treatment. (e) Positive pixel analysis of sections stained for NBR1with Amp HQ showed a persistent increase after 3, 7, and 14 days of DOX treatment. (f) Consistent with IHC results, Western blot analysis showed a decreased intensity of ATG7 and an increased intensity of p62 with DOX-treated groups at days 3, 7, and 14 compared with the no DOX-treated groups.

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