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

Fig. 9.

Fig. 9

Trehalose induces heat shock protein (Hsp)B8 expression and both act in the autophagic clearance of mutant androgen receptor (AR) gene with an abnormally long polyglutamine tract (ARpolyQ). (A) Real-time polymerase chain reaction of HspB8 mRNA expression levels on NSC34 cells expressing AR.Q46 in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours, in basal condition or after treatment with 100 mM of trehalose for 48 hours (°° p < 0.01 vs. AR.Q46−T). (B) Transcriptional activity assay of HspB8 promoter performed on NSC34 cells transfected with promB8 and pRL-TK, in basal condition or after treatment with 100 mM of trehalose for 48 hours (*** p < 0.001 vs. untreated control). (C) Western blot analysis on cell lysates of NSC34 cells expressing AR.Q46 in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours, in basal condition or after treatment with 100 mM of trehalose for 48 hours. β-actin was used to normalize protein loading. (D) 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 100 mM of trehalose for 48 hours (* p < 0.05 vs. AR.Q46+T; ° p < 0.05 vs. AR.Q46+T+trehalose; # p < 0.05 vs. AR.Q46+T+HspB8). (E) Upper insets, Western blot analysis, and lower insets, filter retardation assay, on cell lysates of NSC34 cotransfected with AR.Q46 and a small interfering RNA for endogenous HspB8 (SiRNA-HspB8) or its negative control (scramble). Cells were analyzed in the absence (−T) or in the presence (+T) of 10 nM of T for 48 hours, in basal condition or after treatment with 100 mM of trehalose for 48 hours. α-Tubulin was used to normalize protein loading (# p < 0.05 vs. scramble+T, °° p < 0.01 vs. scramble+T; °° p < 0.01 vs. SiRNA-HspB8+T). Abbreviations: mRNA, messenger RNA; NT, untransfected cells; pCDNA3, empty vector; pRL-TK, renilla luciferase expression vector; promB8, firefly luciferase controlled by HspB8 promoter; SiRNA, small interfering RNA; T, testosterone.