Aluminium Induced Endoplasmic Reticulum Stress Mediated Cell Death in SH-SY5Y Neuroblastoma Cell Line Is Independent of p53
Figure 5
Al(mal)3 induced ER stress in neuroblastoma cells.
(A) Intracellular calcium levels were determined in terms of arbitrary fluorescence units using 5 µM calcium green-1 AM dye at time periods ranging from 30 mins to 300 mins of Al(mal)3 exposure (100 µM–600 µM). Upon binding calcium ions, an increase in fluorescence of the respective dye is observed. (B) Relative quantification (RQ) of CHOP mRNA was done by Real time PCR following Al(mal)3 treatment (100 µM–600 µM) for 24 h. GAPDH was used as endogenous control. (C) Western blot analysis of CHOP and caspase 12 at 24 h of Al(mal)3 treatment (100 µM–600 µM). Band intensities were calculated by densitometry and change in protein expression (Al-treated) was calculated with respect to controls and expressed as fold change in graph. Results were normalized to β-actin. (D) Western blot analysis of Aβ(1–40) following 24 h of Al(mal)3 treatment (100 µM–600 µM) showed oligomers formation at different concentrations of Al(mal)3. (E) Enzymatic activity of caspase 12 was assessed in cell lysates of the SH-SY5Y following treatment with Al(mal)3 (100 µM–600 µM) by the detection of cleavage of substrate ATAD-AFC using fluorimeter (Ex/Em 400/505). The data are represented as means ±SE of three independent experiments.*P<0.05 vs. control; #P<0.05 vs. Al(mal)3 treatment.