Matlab r2016

All data processing was performed off-line using a commercial software package (MATLAB R2016, the MathWorks Inc.). A semi-automatic analysis code was used for cell segmentation and data extraction. N1G4-citrine and Dll1-mCherry cells were segmented separately using the yellow (515nm laser) and red (561nm laser) channels, respectively. For the two-cell assay, TEC was defined as total fluorescence in the yellow channel (green in images shown) found in the area of the Dll1-mCherry cell. Background fluorescence, calculated from pixels outside the segmented area, was subtracted from the TEC signal. Fluorescence signal from the boundary between cells as defined by the overlap of the segmentation of the two cells, was not included in the calculation of TEC. This is because we consider as TEC only N1G4-Citrine within the sender cell and not on the boundary. nTEC was calculated by dividing the TEC by total Dll1-mCherry signal in each time frame. The correlation coefficient between nTEC and contact width and the corresponding p-value was estimated using Pearson correlation analysis. The reporter analysis (Fig. S1N-P) was performed by a semi-automatic tracking protocol in which the total fluorescence in the nucleus was calculated in each frame.

During testing, the compressive force and the shear force were recorded using an integrated 6DOF force transducer. The ROM in FE was obtained using the integrated rotation transducer of the corresponding rotatory channels. NZ was defined as half the total laxity at zero load in the complementary direction, as described by Wilke et al. [16 (link)].
The effects of CORs on the compressive force, shear force, NZ, and ROM in FE were assessed using repeated-measures analysis of variance (ANOVA) with a statistical significance level of 0.05. Prior to the analysis, ROMs of flexion, extension, and NZ of FE were deemed a baseline when the COR was located at the middle point of the horizontal middle plane. Subsequently, ROMs of flexion, extension, and NZ of FE for the 9 CORs were normalized to this baseline, respectively. All statistical analyses were performed with MATLAB R2016 version (MathWorks, Natick, MA, USA).