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

Fig. 4.

Fig. 4

Active autophagy is required to mediate the antiaggregation and/or prodegradative activity of heat shock protein (Hsp)B8 on the androgen receptor (AR) gene with an abnormally long polyglutamine tract (ARpolyQ). (A) Cytofluorimetric analysis performed on NSC34 cells expressing DsRed monomer, 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 mM of 3-methyladenine (3-MA) for 24 hours (** p < 0.01 vs. T-untreated controls; °° p < 0.01 vs. GFP-AR.Q48−T; §§ p < 0.01 vs. GFP-AR.Q48+T; ## p < 0.01 vs. GFP-AR.Q48+T+HspB8). (B) Filter retardation assay performed on NSC34 cells transfected with AR.Q46, 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 mM of 3-MA for 24 hours (* p < 0.05 vs. T-untreated controls; ** p < 0.01 vs. T-untreated controls; °° p < 0.01 vs. AR.Q46−T; § p < 0.05 vs. AR.Q46+T; ˆˆ p < 0.01 vs. AR.Q46+T+HspB8). (C) Western blot analysis on cell lysates of NSC34 cells untrasfected (NT) or expressing AR.Q23 or AR.Q46, in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours. β-actin was used to normalize protein loading. (D) Western blot analysis on cell lysates of 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, in basal condition or after treatment with 10 mM of 3-MA for 24 hours. β-actin was used to normalize protein loading. (E) The histogram represents a quantitative evaluation of LC3-II/LC3-I ratio protein level carried out by densitometric scanning of the blots (3 replicates) (°° p < 0.01 vs. AR.Q46−T; § < 0.05 vs. AR.Q46+T; §§ p < 0.01 vs. AR.Q46+T; # p < 0.05 vs. AR.Q46−T+HspB8; ˆˆ p < 0.005 vs. AR.Q46+T+HspB8). (F) The histogram represents a quantitative evaluation of p62 protein level normalized on actin carried out by densitometric scanning of the blots (3 replicates) (°° p < 0.01 vs. AR.Q46−T; §§ p < 0.01 vs. AR.Q46+T; ** p < 0.01 vs. T-untreated controls; ## p < 0.01 vs. AR.Q46−T+HspB8; ˆˆ p < 0.01 vs. AR.Q46+T+HspB8). Abbreviations: pCDNA3, empty vector; T, testosterone.