Immunofluorescence for autophagic proteins following DMBA treatment.

<p>Immunofluorescence for LC3β (cytoplasmic, green), p62 (cytoplasmic, red) and DAPI (nuclear, blue) at various time points of treatment. (A) At 0 weeks of treatment, there is a low level of punctate LC3β expression and minimal p62 noted, indicating low levels of autophagy in this untreated specimen. (B) After 5 weeks of DMBA treatment, there is evidence of autophagic induction with the increase in punctate, granular LC3β compared to 0 week treatment mice (A). (C) An overlay image with co-expression (LC3β and p62) expressed as orange at the 5 week time point compared to LC3β alone in panel B indicates a mild increase in p62 in addition to LC3β at this time point. (D) With 10 weeks of DMBA treatment, there is significant accumulation of p62 expression and an increase in punctate LC3β indicating autophagic induction without degradation of autophagy-specific substrate p62. (E) At 15 weeks of DMBA treatment, the time of development of high-grade dysplasia, p62 levels begin to decrease with continued evidence of punctate LC3β indicating autophagic induction with the ability to degrade p62. (F) At 20 weeks of treatment, where carcinoma is present, there is again a significant increase in LC3β punctate expression with low levels of p62. All images are acquired at 20x magnification.