Figure 8.

Downregulation of CNPY2 reduces the viability of cultured striatal neurons. Striatal cells were cultured in 6 well plates for immunoblotting or in 96 well plates for cell viability assays. (A) Striatal cells were transfected with shRNA-CNPY2 plasmid or scrambled shRNA for 24 h, and 1 or 2.5 μg/mL tunicamycin (T) was then added to half of the cells for 16 h. Cell viability was then determined using 8 wells for each treatment and the experiments were repeated. Downregulation of CNPY2 rendered the striatal cell more vulnerable to tunicamycin treatment compared with controls. Values are means ± SD, n = 4. ^**p < 0.01 for 1 μg/mL T vs. C, ^***p < 0.001 for 2.5 μg/mL vs. C in both groups, and ^**p < 0.01 for sh-CNPY2 + 2.5 μg/mL T vs. C + 2.5 μg/mL T. (B) Immunoblots. Cells were treated with 2.5 μg/mL tunicamycin for 24 h. Levels of CNPY2 were reduced after shRNA-CNPY2 expression. Control cells received scrambled shRNA. Splicing of XBP1 (XBP1s) and CHOP induction were changed in CNPY2 downregulated cells. GAPDH was used as a control for blotting. (C) Quantification. XBP1s induced by tunicamycin was reduced after CNPY2 downregulation, whereas that of CHOP was further increased. There was no significant effect on induction of phosphorylated eIF2α level by CNPY2 downregulation. Values are means ± SD, n = 3. ^**p < 0.01 or ^*p < 0.05 for shCNPY2 + Tun vs. Tun.