Microcal origin 6

Morphological properties of MAs from NT rats and SHRs as well as NT MAs treated with NTC miR or miR153 were studied using Pressure Servo System PS/200 (Living Systems Instrumentations, Burlington, VT, USA). The structural properties of the segments and responses to the Kv7 activator, S1 (NeuroSearch A/S, Ballerup, Denmark) were recorded and acquired with DMK 41AU02 Monochrome Industrial Camera (Imaging Source, Bremen, Germany) hosted by a PC running MyoVIEW II software (Danish Myo Technology, Aarhus, Denmark). Data were analysed using MyoVIEW II and MicroCal Origin 6.0 (MicroCal Software, Northampton, MA, USA). Isometric tension was recorded on NT MAs or MCAs transfected with either mir153 or NTC miR in a wire myograph (Danish Myo Technology). Mesenteric vessels were pre-constricted with 1 µM U46619 (Sigma Aldrich) and a concentration effect curve to the Kv7.2–7.5 activators ML213 (HelloBio, Bristol, UK), ICA-069673 (HelloBio), the β-adrenoceptor agonist isoprenaline (Sigma Aldrich), or the Kv7.1-specific activator RL-3 (Tocris, Bristol, UK) as well as a relaxation response to 1 µM nicardipine (Sigma Aldrich) were obtained. Relaxant responses to S1 or ML213 were also assessed in MCAs transfected with miR153 or NTC and pre-constricted with 100 nM U46619. Data were recorded and analysed using LabChart® 7 (ADInstruments, Dunedin, New Zealand).

Spectra of the samples were obtained using an FT-Raman Spectrometer (RFS 100/S; Bruker Inc. Karlsruhe, Germany). To excite the spectra, the defocused 1064.1 nm line of an Nd:YAG laser source was used. The maximum incident laser power on the sample surface was approximately 150 mW and the spectrum resolution was 4 cm^-1.
The samples were positioned in the sample holder compartment and an IR352 lens collected radiation scattered through 90° on the dentin surface. For each sample, one spectrum was collected at a central point on the cervical dentin root. In order to obtain a good signal to noise ratio, 100 scans were co-added for each spectra. Five spectra were obtained in each group.
The changes in the organic dentin components were analyzed by comparing the integrated areas of the Raman peak centered at 2940 cm¹ . The integrated areas of the peaks were calculated with the software Microcal Origin 6.0 (Microcal Software, Inc., Northampton, MA, USA).

All data analysis was performed using GraphPad Instat Software Inc., USA. Blastocyst rate and hatching rate is represented as percentage data and was evaluated by Chi Square statistics. The different variables are expressed as Mean ± SEM (Standard error of mean). Student’s t-test was applied to normally distributed data and Mann-Whitney U-test was applied to data that did not conform to normal distribution. P value < 0.05 was considered statistically significant. The graphs were plotted using Microcal Origin 6.0 (USA).

The statistical significance of data collected by EM analyses was determined using a Student's t test (Microcal Origin^® 6.0; Microcal Software, Inc., Northampton, MA), while statistical significance of percentage values was investigated using a chi‐squared test (Microsoft^® Office Excel^® 2007; Microsoft Corporation). Statistical analysis of morphometric, densitometric, and gene expression datasets was performed with GraphPad Prism v5.0 software (GraphPad Software, Inc., La Jolla, CA); statistical significance of average numbers was determined using Wilcoxon matched pairs test. Values of P < 0.05 were considered significant.

The initial velocity of ATP hydrolysis was calculated at different ATP concentrations from the slope of the linear portion of the NADH absorbance decay [48 (link)]. The observed sigmoid dependence on the velocity of ATP hydrolysis with the substrate concentration was analyzed fitting the data to the Hill equation (Equation (1)) by nonlinear regression using the iterative software Microcal Origin 6.0^® (Northampton, MA, USA).
$v=\frac{V_{\max }\cdot S^n}{S_{0.5}^n+S^n}$
where v is the initial rate, V_max is the maximum velocity of ATP hydrolysis, S is the ATP concentration, S_0.5 is the ATP concentration when v = 0.5V_max, and n is the Hill number.
The H⁺-ATPase steady-state fluorescence intensity at λ of 332 nm was plotted against nucleotide concentration (AMP-PCP and ADP). The fluorescence data were fitted to Equation (2) by nonlinear regression [22 (link)]. Equation (2) describes the binding of the substrate considering the existence of simple symmetric cooperativity in the protein [50 (link)].
$\left(F_0-F\right)=\frac{\Delta F_{\max }\cdot S^n}{K_d^n+S^n}$
where (F₀ − F) represents the quenching of steady-state fluorescence at λ of 332 nm at a given nucleotide concentration S. ΔF_max is the maximum change in fluorescence intensity generated by substrate binding; n is the number of binding sites involved in the interacting unit; and K_d is the average dissociation constant of the nucleotide from the binding site [50 (link)].

Data are average results obtained by means of five replicates and independent experiments and are presented as average ± standard errors of the mean (SEM). Data were subjected to analysis of variance (ANOVA) with significant differences between means identified by GLM procedures. Results are given in the text as probability values, with p < 0.05 adopted as the criterion of significance. The LD₅₀ calculated by PROBIT analysis based on the percentage of inhibition obtained at each concentration of the samples. LD₅₀ is the concentration that produces 50% mortality. Complete statistical analysis was performed by means of the Micro-Cal Origin 6.1 statistical and graphs PC program.