Figure 5. Lead treatment causes a disruption of cellular calcium balance. Pre-treatment of N27 cells with 1 mM N-acetylcysteine (NAC) for 1 hour followed by a 48-hours lead-exposure was used to investigate the impact on mitochondrial ROS (A) and mitochondrial depolarisation (MMP) (B) N27 cells treated with lead for 48-hours were tested for cytoplasmic (C) and ER calcium levels (D) using thapsigargin (Tg) and Rhod-2 staining followed by flow cytometry analysis (D, a and b). Calcineurin transcript levels 48-hours post-lead exposure were investigated using qRT-PCR (E). Pre-treatment of N27 cells with 5μM BAPTA-AM for 1 hour followed by a 48-hours lead-exposure was used to investigate the impact of calcium on mitochondrial depolarisation (F) and cell death (G) following lead treatment. (H) Western blot of BAP31 protein levels after 48-hours lead treatment. (I) Western blot analysis of BAP31 levels in shBAP31 N27 cells. N27 cells with stable downregulation of BAP31 (shBAP31) were used to investigate the involvement of BAP31 in lead-induced mitochondrial superoxide generation (J), mitochondrial depolarisation (MMP) (K) and activation of caspases 3/7 (L) and 8 (M). *P<0.05, **P<0.01, ***P<0.001, n=3; mean ± SE.