Supplementary MaterialsSupplement 1. cells (7.61 vs. 4.65 protrusions/cell), GTM protrusions were significantly longer (12.1 m vs. 9.76 m). Live-cell imaging shown the GTM actin cytoskeleton was less dynamic, and vesicle transfer between cells was significantly slower than NTM cells. Furthermore, rearrangement of the actin cortex adjacent to the TNT may influence TNT formation. Myosin-X immunostaining was punctate and disorganized in GTM cells and cells compared to age-matched NTM settings. Conclusions Together, our data demonstrate that GTM cells have phenotypic and practical variations in their TNTs. Significantly slower vesicle transfer via TNTs in GTM cells may delay the timely propagation of cellular signals when pressures become elevated in glaucoma. bioparticles (ThermoFisher) were added to each well of a 6-well plate comprising GTM or NTM cells. The plate was placed in the Incucyte Focus instrument STATI2 (Essen Bioscience, Ann Arbor, MI, USA), and each well was imaged every quarter-hour for 18 hours by using the phase and reddish fluorescence channels. Fluorescence at each time point was measured using open-source FIJI software (http://fiji.sc/Fiji). Data are from three technical replicates of 3 GTM and NTM cell strains. Cellular Senescence Assay Cellular senescence was measured using a -galactosidase staining Omniscan cell signaling kit (Cell Signaling Systems, Danvers, MA, USA) following a manufacturer’s Omniscan cell signaling directions. Images were acquired using a BX51 microscope (Olympus, Waltham, MA, USA) equipped with a DC500 digital camera (Leica, Deerfield, IL, USA). FIJI was used to measure average pixel intensity for three images from NTM and GTM cell Omniscan cell signaling strains (= 3 each). Data were averaged, and significance was determined using a 1-way ANOVA. Immunostaining and Measurement of Cell Size and Cellular Protrusions For immunostaining experiments, NTM and GTM cell strains (2 105 cells/mL) were cultured on collagen I-coated BioFlex plates (FlexCell International Corp, Burlington, NC, USA) for 16 hours. This allowed the cells to adhere, but the cells were not too confluent. Cells were fixed in 4% paraformaldehyde and incubated with CD44 main antibody (rat monoclonal anti-CD44, clone IM-7; Stem Cell Systems, Vancouver, BC, Canada) and Alexa-fluor 594-conjugated donkey anti-rat secondary antibody (ThermoFisher). Coverslips were mounted in ProlongGold mounting medium comprising 4,6-diamidino-2-phenylindole (DAPI; ThermoFisher) and visualized using a Fluoview FV1000 confocal microscope (Olympus). Z-stacks were placed 0.5 m above and 0.5 m below the fluorescent signal to ensure that the entire cell depth was captured. The area (m2) and volume (m3) of NTM and GTM cells were determined from z-stacks using the surfaces module Imaris software (Bitplane, Concord, MA, USA). Partial cells in each image were not counted. If the cells were touching, they were Omniscan cell signaling manually separated in the software, and if indeed they cannot become separated quickly, those images were discarded then. To gauge the accurate quantity and amount of filopodia, the filaments module was used. The beginning of a protrusion in the cell surface area and end from the filaments had been by hand assigned in the program. To gauge the colocalization of cortactin and Myo10, the coloc module was utilized to make Omniscan cell signaling a Pearson’s worth, which quantitatively actions the amount of overlap of fluorescent indicators acquired in various fluorescent stations.39 Colocalization was categorized as quite strong (0.88C1.0), strong (0.61C0.87), average (0.4C0.6), weak (0.13C0.39), and incredibly weak (0C0.12).40 Actin pressure fiber diameters were measured from confocal pictures through the use of ImageJ. Vesicle Transfer Assay The real amount of vesicles transferred was quantitated utilizing a vesicle transfer assay.20,41 Briefly, one flask of confluent TM cells was trypsinized, and fifty percent was labeled with Vybrant DiO dye (488 nm), as the spouse was labeled with DiD dye.