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. 2013 Nov;34(11):2585–2603. doi: 10.1016/j.neurobiolaging.2013.05.026

Fig. 6.

Fig. 6

Autophagic flux is impaired by the mutant androgen receptor (AR) gene with an abnormally long polyglutamine tract (ARpolyQ) and restored by heat shock protein (Hsp)B8. (A) Cytofluorimetric analysis performed on NSC34 cells expressing mRFP-LC3, GFP-AR.Q48, pCDNA3, or HspB8 in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours, in basal condition or after treatment with 10 μM of MG132 or 10 mM of 3-methyladenine (3-MA) for 24 hours (°° p < 0.01 vs. GFP-AR.Q48−T; §§ p < 0.01 vs. GFP-AR.Q48+T). (B) Real-time polymerase chain reaction (PCR) on LC3B mRNA expression levels on NSC34 cells expressing AR.Q46, pCDNA3, or HspB8 in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours (°° p < 0.01 vs. AR.Q46−T; * p < 0.05 vs. AR.Q46+T). (C) Top insets, Western blot analysis of cell lysates of NSC34 cells expressing AR.Q46, pCDNA3, or HspB8 and shRNA against LC3B or shRNA scrambled control in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours. α-Tubulin was used to normalize protein loading. Bottom inset, real time PCR on LC3B mRNA levels on NSC34 cells expressing shRNA-LC3B or shRNA-control. Data (expressed as fold changes) have been normalized to the amount of GAPDH mRNA, and are expressed as relative to the levels determined in shRNA-control transfected cells, which are taken as internal reference. Data are mean ± SD of 4 independent replicates. (D) Cytofluorimetric analysis performed on NSC34 expressing mCherry-p62, GFP-AR.Q48, pCDNA3, or HspB8 in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours, in basal condition or after treatment with 10 μM of MG132 or 10 mM of 3-MA for 24 hours (*** p < 0.001 vs. T-untreated controls; °° p < 0.01 vs. GFP-AR.Q48−T; § p < 0.05 vs. GFP-AR.Q48+T; §§§ p < 0.001 vs. GFP-AR.Q48+T). (E) Real-time PCR on p62 mRNA expression levels on NSC34 cells expressing AR.Q46, pCDNA3, or HspB8 in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours (°° p < 0.01 vs. AR.Q46−T; * p < 0.05 vs. AR.Q46−T+HspB8). (F) High resolution immunofluorescence analysis on NSC34 cells transfected with GFP-AR.Q48 in the absence (−T) or in the presence (+T) of 10 nM of T or 100 nM of cyproterone acetate or 100 nM of casodex for 48 hours. Nuclei were stained with DAPI (blue). Images were obtained at magnification ×63 (scale bar, 10 μm). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MG132, Z-leu-leu-leu-al; mRFP-LC3, monomeric red fluorescent protein-LC3; mRNA, messenger RNA; pCDNA3, empty vector; shRNA, short hairpin RNA; T, testosterone.