Figure 2

Stimulation with PRE084 counteracts the deleterious effects caused by N-terminal mutant huntingtin proteins. PC6.2 cells expressing 18Q- and 120Q-containing N-terminal huntingtin fragment proteins were left untreated or treated with the Sig-1R agonist, PRE084 as described in the Materials and Methods. (a) Cell viability was assessed after 24 h by the MTT assays as described in the Materials and Methods. PRE084 reduced cell death induced by the 120Q mutant huntingtin. Values represent means±S.D., n=3. *P<0.05 for 120Q versus C and for PRE084+120Q versus 120Q. (b) Cell death was assessed by counting number of apoptotic nuclei in 18Q and 120Q-huntingtin-fragment protein-expressing cells incubated for 24 h and 48 h without or with 0.3 μM PRE084. Cell death increased in cells transfected with the 120Q huntingtin and this was reduced by PRE084. Values represent means±S.D., n=3. **P<0.01 for 120Q versus 18Q at 24 h and *P<0.05 at 48 h. (c and d) Immunoblot and quantification. Expression of 120Q-huntingtin-induced cleaved caspase-3 (17 kDa band) and this was reduced by addition of 0.3 μM PRE084. The cleavage of PARP (89 kDa band) a downstream substrate of caspase-3 was also decreased by PRE084. β-actin was used as control. Values represent means±S.D., n=3. **P< 0.01 for 120Q versus C and for PRE084+120Q versus 120Q. (e) Activity of caspase-3 was measured as described in the Materials and methods. Note an increase in cells expressing 120Q mutant huntingtin that as reduced by 0.3 μM PRE084. Values represent means±S.D., n=3. **P<0.01 for 120Q versus C and for PRE084+120Q versus 120Q. (f and g) Immunoblot and quantification. Expression of 120Q-huntingtin-induced cleaved caspase-12 (36 kDa band) and this was reduced by addition of 0.3 μM PRE084. β-actin was used as control. Values represent means±S.D., n=3. *P< 0.05 for 120Q versus C