Figure 5.

A comparison of the effects of stabilizing and destabilizing osmolytes versus that of heat shock in alleviating the diffuse mHtt dose-dependent repression of CRE-firefly luciferase reporter gene. The 14A2.6 line of PC12 cells were transfected with the CRE-firefly luciferase reporter DNA along with the internal control CMV-driven humanized Renilla luciferase reporter DNA as described⁹. After transfection, cells were plated at t = 0 into a 96 well plate and ponasterone (0–5 µM) was added to designated wells to final concentrations shown. Osmolytes (120 mM) were added at t = 24 h and incubation continued at 37 °C. Heat shock of cells was for 2 h at 42 °C at t = 24 h followed by recovery at 37 °C. The CRE-reporter gene was induced by the addition of 1 mM dibutyryl cAMP at t = 48 h and incubation for an additional 6 h at 37 °C. Reporter gene activity was assayed using the Dual-Glo reporter gene assay kit from Promega Inc. Results on the firefly luciferase reporter gene activity was normalized against that of the Renilla luciferase relative to the ratio of 1 for the un-induced control (basal; without PA or dibutyryl cAMP induction). Result is the average from three separate experiment each with 8 independent determinations. Probability of difference P > 0.05 is defined as not significant, between 0.01 and 0.05 is significant (*), < 0.01 is very significant (**), and < 0.001 is extremely significant (***).