Fig. 5. Enhanced ER Ca²⁺ loss in β-cells is due to presenilin-1. (A) Representative gel showing detection of mRNA levels of presenilin-1 in HeLa, EA.hy926, INS-1 and MIN-6 cells via standard PCR using gene and species specific primers (n = 3). (B) Quantification of mRNA expression levels of presenilin-1 in HeLa, EA.hy926, INS-1 and MIN-6 cells compared to mRNA levels of the housekeeping gene GAPDH detected with qPCR (n = 3). Bar charts indicate mean + SEM. (C) Quantification of knockdown efficiency of presenilin-1 in HeLa, EA.hy926, INS-1 and MIN-6 cells compared to the housekeeping gene GAPDH using real-time PCR (n = 3). (D,E) Evaluation of the influence of presenilin-1 knockdown (siPS1) and over-expression (PS1 OE) on ER Ca²⁺ leakage - depicted as percentage of the initial ER Ca²⁺ content in (D) INS-1 and (E) MIN-6 under control conditions (black lines), upon knockdown of presenilin-1 (red lines) or over-expression of presenilin-1 (green lines) after incubation under Ca²⁺-free conditions. ER Ca²⁺ stores were depleted at the indicated time by treating the cells with 15 µM of the SERCA inhibitor BHQ and 100 µM carbachol. Points represent the mean values ± SEM (n ≥ 6). (F) Corresponding statistics for panels D and E showing the % of ER Ca²⁺ content in INS-cells (upper panel) and MIN-6 cells (lower panel) after 20 min incubation in Ca²⁺-free buffer and after ER Ca²⁺ stores were depleted by 100 µM Cch and 15 µM BHQ. Respective 1 min values were set to 100%. Bars represent mean ± SEM (n ≥ 6). *p<0.05 versus respective 1 min control or as indicated in the graph using one-way ANOVA. (G) Percentage of ER Ca²⁺ content in INS-1 cells after 20 min of incubation under Ca²⁺-free conditions either under control conditions or after knockdown of the indicated types of IP₃R. ER stores were depleted after 20 min by applying 0.2 µM of ionomycin together with the SERCA inhibitor BHQ (15 µM). In each graph the 1 min control value was set to 100% (n ≥ 5). *p<0.05 versus respective 1 min control or as indicated in the graph using one-way ANOVA. (H) Percentage of ER Ca²⁺ content in INS-1 cells after 20 min of incubation under Ca²⁺-free conditions either under control conditions or after knockdown of the indicated types of IP₃R. ER stores were depleted after 20 min by applying 0.2 µM of ionomycin together with the SERCA inhibitor BHQ (15 µM). In each graph the 1 min control value was set to 100% (n ≥ 5). *p<0.05 versus respective 1 min control or as indicated in the graph using one-way ANOVA.