Prism ver 6

Statistical analyses were performed using a statistics software (Prism ver. 6, GraphPad software Inc., San Diego, CA, USA). Values are expressed as the mean ± SD or median and interquartile range (25%–75%). The respiratory function data were analyzed by a Friedman’s test. For CT and bone analysis, values in the stimulation group were compared with the sham stimulation group using Student’s t-test or Mann–Whitney U-test. One-tailed tests were used for analysis of our main hypothesis that CT concentration and BMD in the stimulation group would be higher than that in the sham stimulation group. Otherwise, two-tailed tests were used. Statistical significance level was set at 5%.

To measure the muscular strength of male and female Sil1^Gt and wild-type mice, we performed an inverted screen test (Deacon, 2013 (link)) using a 12″×8″ screen (0.5″ metal grid) that was surrounded by 9″ plexiglass walls to prevent mice from climbing onto the top of the screen during the assay. A pillow was placed below the apparatus to cushion the fall. Each mouse was placed on the screen, allowed to acclimatize for a few seconds, and the screen was rotated 180° to position the mouse upside down, at which point timing began. The length of time that each mouse was able to hold on to the screen was recorded with an assay maximum limit of 120 s. Each mouse was subjected to a total of three trials successively and the average was determined. Mean times for wild-type and Sil1^Gt mice of each age group were statistically compared using Prism Ver. 6 (GraphPad Software, La Jolla, CA) by Mann–Whitney tests (unpaired; two-tailed).

two-way ANOVA with correction for multiple comparison using the Sidak method was conducted to determine if GPCRs can activate G proteins and the rank order of G protein selectivity with GraphPad Prism Ver. 6. Only statistically significant values are reported. Values represent means ± SEM from three independent experiments each performed with at least three replicates.

Multiple t tests with correction for multiple comparison using the Holm–Sidak method was conducted to determine the effect of RGS on the deactivation rates of Gα subunits with GraphPad Prism Ver. 6. Only statistically significant values are plotted. Values represent means ± SEM from three independent experiments each performed with three replicates.

We performed the Shapiro–Wilk normality test and Brown-Forsythe equal variance test. If the data passed the normality test and equal variance test, we then compared groups by using Student’s t test. If the data failed the equal variance test, we applied log transformation. To compare among groups, we performed one-way ANOVA test with Holm-Sidak pairwise multiple post hoc comparisons or one-way ANOVA rank test (SigmaPlot ver. 14, Systat Software). In all cases, we used two-tailed tests with significant difference set at P < 0.05. Data were displayed as mean ± SD and figures in scatterplots (Prism ver. 6, GraphPad).

The neuroblastoma cells (SH-SY5Y) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). SH-SY5Y cells were maintained in Dulbecco’s modified eagle media (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% kanamycin, and 1% penicillin. Cell cultures were maintained at 37 °C in 5% CO₂ and passaged once per week. The SH-SY5Y cells were subcultured into a 96-well plate at 2 × 10⁵ cells/well and incubated for 24 h. After incubation, the cells were treated with plant extracts at different concentrations and incubated for another 72 h. The medium was removed, and the wells were washed with phosphate-buffered saline (PBS). Fresh medium (100 μL) was added and incubated for another 30 min. After incubation, CellTiter-Glo^® luminescent reagent (100 μL; Promega, Madison, WI, USA) was added, and the luminescence was measured using a PerkinElmer Victor-3^® multi-plate reader (PerkinElmer, Waltham, MA, USA) [48 (link)]. The 50% inhibitive concentrations of the plant extracts were calculated using a nonlinear regression curve fit (GraphPad Prism ver. 6, San Diego, CA, USA). The values representing cell viability were expressed as means ± standard deviation (SD) of three trial experiments.