Igor software

Data analysis is performed using Igor software (Wavemetrics) and Matlab (MathWorks). Statistical tests are performed with Matlab using, if not otherwise indicated, a Student's t-test in comparison of two datasets, a statistical test comparing the slope of a least-squares linear best fit line to 0, or a least-squares regression to an exponential function when such a trend is expected (subthreshold and action potential attenuation). Statistical tests are two-tailed unless a reason is explicitly stated to expect a directional relationship between two datasets.
To estimate the width of dendritic plateau potentials in the apical dendrite with long dendritic current injection, we determine the longest depolarization sustained at 20% or more above the baseline level (defined as the most hyperpolarized membrane potential during the dendritic current injection). This includes the effects of backpropagating APs as well as their interplay with the dendritic depolarization.

The adhesion experiments were performed as described previously14 (link). Briefly, the monocytes were subjected to laminar flow through a channel (2 mm width and 0.1 mm height) inserted into a well of a 24-well chamber. The shear rate was ~10 s⁻¹; thus, the cells attached to the surface were subjected to a viscous force on the order of 7 pN23 (link). Images were acquired with a video camera (HyperHAD; Sony France, Clichy, France) attached to an Olympus IX 50 inverted microscope with a 10× objective set within a closed box maintained at 37 °C. The pixel size was 1 × 1 μm². Sequences, typically 2 min in duration, were digitized and DivX compressed with a Win-TV digitizer (Hauppauge, France) for subsequent analysis. The films were decomposed into frames using ImageJ software (1.48v; NIH, USA). Subsequently, the TrajFSerie program (written in Java) used these tables to create a file containing the trajectory of each cell. This file was processed with Igor software (Wavemetrics, EUA) to detect and measure the duration of the stops of each cell. The numbers of detectable arrests (remaining for 200 ms or longer) and the numbers of permanent arrests (i.e., arrests lasting 2 min) were thus determined.

SRCD spectra were recorded on the DISCO beamline36 (link) at synchrotron SOLEIL (Saint-Aubin, France). The experimental setup was calibrated for magnitude and polarization with a 6.1 mg/ml D-10-camphorsulfonic acid solution. Two μl of WT mt-AspRS or mutants solution at 10 mg/ml in 100 mM potassium phosphate pH 7.5, 50 mM KCl, 10% (v/v) glycerol and 1 mM TCEP were transferred in a CaF₂ cuvette with an optical path of 8 μm. Three spectra from 170 to 280 nm were recorded at temperatures ranging from 24 to 80 °C with 4 °C steps. They were averaged, solvent baseline subtracted, scaled and normalized to molar ellipticities using CDtool37 (link). Protein stability was determined by plotting the variation of the CD minimum at 209 nm as a function of temperature. IGOR software (WaveMetrics) was used to model this variation by a sigmoid and the temperature corresponding to the maximum of its derivative defined a structural transition or melting temperature (Tm). Secondary structure content was estimated from SRCD spectra collected at 24 °C using CONTINLL in Dichroweb and SELCON3 in CDPro using the SP175 reference data set38 (link)39 (link)40 (link). A data cutoff at 180 nm was applied based on photomultiplier high-tension viability. Values were compared to those in the X-ray structure of the WT human mt-AspRS (PDBid: 4AH6).

Statistical analysis was performed using Fitmaster (version 2.69; HEKA, Germany) and Igor software (version 6.34; WaveMetrics, USA). The p-values were calculated using Microsoft Excel version 14.1.2 using the two-tailed paired Student’s T-test. Data were normalized to cell size and expressed as current density (pA/pF), or normalized to the time point right before application (I/I_[time]s). Data were displayed as mean ± standard error of mean (s.e.m.). Dose-response fits were assessed using the fit function f(x) = ([Maximum current] *(1/(1 + ([Estimated IC₅₀]/x)^[Hill coefficient])). The IC₅₀ or EC₅₀ values calculated with the dose-response curve function were reported with ± standard deviation (S.D.).