Androgen-mediated cellular senescence is linked to autophagy activity. To examine the association between the androgen-mediated induction of SA β-gal activity as a specific marker for cellular senescence and autophagy the conversion of LC3 was analyzed after 3 d incubation of LNCaP cells with SAL or LAL. A. The conversion of LC3 I to LC3 II as a marker of autophagy activity was detected by Western blotting experiments. LNCaP cells were treated with the Src tyrosine kinase inhibitor PP2 (1 μM) or the mTOR inhibitor rapamycin (1 nM) in addition to SAL and the conversion of the autophagy marker from LC3 I to LC3 II was detected via Western blotting. Quantification of the bands was performed by the Labimage D1 program. The expression levels of LC3 I and II were normalized to the band intensities of the loading untreated sample control β-actin that was set as 1. The ratios of these values are indicated below. B. The puncated pattern of LC3 is indicative for autophagy activity and was analyzed by using GFP-LC3. LNCaP cells were transfected with GFP-LC3 and treated with the indicated compounds for the indicated period. The distribution of GFP-LC3 and formation of puncated pattern was analyzed by fluorescence microscopy. C. The up-regulation of p14 gene expression by SAL is inhibited by an inhibitor of PI3K and autophagy. qRT-PCR of LNCaP cells that were treated with the indicated time periods with and without SAL and 3-MA as described in Figure 3B.