Figure 6.

Evaluation of [Ca²⁺] ER with ER-targeted cameleon (D1ER) probe and evaluation of intracellular reactive oxygen species (ROS) level in stressed SH-SY5Y cells stably expressing URG7 protein. (A) The steady state D1ER FRET signal (ratio 530/470 nm) in SH-SY5Y cells expressing either the empty vector (pLV) or URG-7 vector (pLV-URG7) treated or not with tunicamycin (TN) is depicted in pseudocolor. The histogram compares changes in netFRET in control cells in both basal conditions (pLV; n = 31 cells) and after TN treatment (pLV + TN; n = 46 cells) and in URG-7-expressing cells in both basal conditions (pLV-URG7; n = 62 cells) or after TN treatment (pLV-URG7 + TN, n = 71 cells). Data are expressed as means ± Standard Error Measurement (SEM). Statistical analysis was performed on three independent experiments and significance calculated by ANOVA. **p < 0.01 pLV + TN vs. pLV. (B) Representative Western blotting and (C) densitometric analysis performed in SH-SY5Y clones treated with 3 μg/mL of TN for 15 h. β-actin was used as the loading control. Results, expressed as means ± Standard Error Measurement (SEM) of three independent experiments, are shown as fold change compared with the control value (mock). Statistical significance was evaluated using GraphPad Prism 8.4.2 software (unpaired, two-tailed t-test, * p< 0.05, ** p < 0.01,). (D) SH-SY5Y cells stably transfected with pLV and pLV-URG7 were treated with TN for 3, 6 and 15 h and the levels of intracellular ROS were detected using DCFH-DA fluorescent probe by FACScan flow cytometry. All data, expressed as means ± Standard Error Measurement (SEM) of three independent experiments, are shown as % of the control value (mock) and statistical significance was evaluated using GraphPad Prism 8.4.2 software (unpaired, two-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).