Nis elements software version 3

Fluorescence images of the 2D cultures were taken by a Zeiss Axiovert A1 inverted fluorescence microscope (Carl Zeiss, Jena, Germany) equipped with an AxioCam MRm CCD camera and a, LED excitation light source (Thorlabs, Newton, NJ, USA). Individual cells were counted by using the Zen 2 counting mode (Carl Zeiss). Fluorescence images and intensities of the 3D/ECM hiDPSCs and hpDPSCs immunostained cultures were taken and quantified by stitching sliced images along the z-axis (motorized inverted Eclipse Ti-E epifluorescence microscope; Nikon Instruments Inc., Melville, NY, USA) by using the NIS-Elements software version 3.0 (Nikon Instruments Inc). Cross-sectional analysis of the developing structures was performed by Z-stacking images in each channel into a single image representing both fluorescence channels. The images were collected by laser-scanning confocal microscopy (LCSM; 3.5 µm intervals, two fluorescence channels; Leica Microsystems Inc., Buffalo Grove, IL, USA). To measure the thickness and diameter, 25 to 30 constructs/microtissues were observed randomly per each experimental group. Thicknesses of constructs were measured along both x–z and y–z planes using Leica Application Suite Advanced Fluorescence (LAS AF) software (Leica Microsystems Inc). The 3D structures were created from stacking images obtained by LCSM, and analyzed by Image J software (V.1.48, NIH).

DNA uptake assays were performed as similarly described for the analysis of natural competence among X. fastidiosa strains (see SI Materials and Methods in S1 Text). Briefly, recipient cells were suspended in PD3 broth to OD_600nm = 0.6, spotted onto PD3 agar plates and grown for three days at 28°C. Then, 1 μg of fluorescently labeled pAX1-Cm plasmid (10-μl volume), labeled using the Label IT Nucleic Acid Labeling kit, Cy3 (Mirus Bio LLC), was added on top of cells, air-dried, and incubated at 28°C for another 24 h. After, cells were harvested in 150 μl of PD3 broth, and a 50 μl aliquot was treated with 10 units of DNase I (New England Biolabs) for 10 minutes at 37°C to degrade the remaining extracellular DNA, and cells were observed using a 100× oil immersion objective in a Nikon Eclipse Ti inverted microscope (Nikon). Image acquisition was performed using a Nikon DS-Q1 digital camera (Nikon) controlled by the NIS-Elements software version 3.0 (Nikon), which was also used to create merged fluorescent images. To detect Cy3, an excitation wavelength of 590 nm was used (tetramethyl rhodamine isothiocyanate; TRITC filter). The proportion of cells that acquired extracellular DNA was calculated as the percentage of cells with fluorescent DNA foci to cells without fluorescent DNA foci. Experiments were performed at least three times independently.