Origin 8

To account for any differences in cell density the amount of Ca²⁺ released at each concentration of 4-cmc was normalized for each assay by expressing Ca²⁺ release as a percentage of the total Ca²⁺ released with 1000 μM 4-cmc. A minimum of eight replicates was used for each RyR1 variant including five replicates for each concentration of 4-cmc. The data were pooled and the mean±standard error of the mean (SEM) was calculated for each value of 4-cmc used. A sigmoidal curve was fitted for each dataset using the Sigmoidal Logistic function, type 3 within OriginLab Origin 8 software and the half maximal effective concentration (EC₅₀) values were calculated from individual sigmoidal curves. Statistical significance of the difference between the EC₅₀ values for each of the variants and the normal (wild-type control) variant was calculated in the form of p-values using an unpaired Student’s t-test using OriginLab Origin 8 software. An alpha level of 0.0017 was used for all statistical tests according to the Bonferroni correction for 99% significance.

The amount of Ca²⁺ release at each concentration of 4-CmC was normalized to account for any differences in cell density for each assay and calculated as a percentage of the total Ca²⁺ released with 1000 µM 4-CmC. A minimum of 8 analytical replicates were carried out for each construct, the results were pooled and a sigmoidal curve fitted for each data set using OriginLab Origin 8 software. Results are presented as the mean ± standard error of the mean (SEM) for each value of 4-CmC used. The concentration of agonist required for half maximal fluorescence change (EC50) was calculated from curves fitted to individual replicates and represented as mean ± SEM. The statistical significance of each EC50 value was determined using the unpaired Student's t-test with OriginLab Origin 8 software and represented as a p-value with respect to WT and a Bonferroni correction was carried out to confirm statistical significance.

Lipid extracts were analyzed with a flow-injection system using a flowrate of 1 μl/min and 5 μl sample injection. Negative and positive ion mode spectra were acquired with a LTQ-Orbitrap XL equipped with a 1200 microLC system and a Nanomate Triversa utilizing 5-μm ID ESI-chips. In the negative mode, PI, PE, PE-O, lysophosphatidylethanolamine (LPE), PC, CerPE, phosphatidylserine (PS), and phosphatidylglycerol (PG) were identified according to their accurate mass as described earlier (Schwudke et al., 2011 (link)). For PS, the specific neutral loss of 87 Da was monitored in the linear ion trap and used for quantification. In the positive ion mode Sph 14:1, ceramides and hex-ceramides were monitored with MS³ in the linear ion trap using the long chain base-related fragment ions. All MS³ for quantifying sphingolipids were analyzed using Xcalibur software while all other analyses were performed using LipidXplorer (Herzog et al., 2011 (link)).
For single and pooled brains, absolute levels of individual lipid species were summed up to arrive at lipid class quantities. At least five biological replicates were used for the analyses. GraphPad Prism (GraphPad Software, USA) and Origin 8.1 (OriginLab, USA) were used to for graphical representation and Origin 8.1 for analysis of variance (ANOVA) coupled with post-hoc Tukey test.