Rangamani P, et al. phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular business to modulate signaling that enables phenotypic changes. cat # 3501Nogo-A/Reticulon-4IF 1:100Cell signaling, cat # ab47085-tubulinIF 1:100Cell signaling, cat # 2144AIF mitochondrial marker (D39D2)IF 1:100Cell signaling, cat # 5318EEA1early endosome marker (C45B10)IF 1:100Cell signaling, cat # 3288RCAS1 (D2B6N) Golgi markerIF 1:100Cell signaling, cat # 9091Muscarinic acetylcholine receptor Rabbit polyclonal to NUDT7 3 (M3R)IF 1:100Abcam, cat # ab126168NFATc1 antibodyIF 1:100Abcam, cat # ab2722SRF antibodyIF 1:100Cell signaling, cat # 4261MyocardinIF 1:100Abcam, cat # 22073 Open in a separate windows Airyscan imaging of live cells VSMC conforming in the 3D biochips were simultaneously labeled with 1?M CellMask Plasma Membrane tracker (Life Technologies), 1?M CellMask ER marker (BODIPY TR Glibenclamide), in HBSS buffer supplemented with 1% Pyruvate, 1% HEPES and 1?mM Trolox, for 5?min at room temperature. Images were acquired using Zeiss LSM 880 using Airyscan super-resolution imaging equipped with 63?x 1.4 Plan-Apochromat Oil objective lens at 30?C. Z-stacks with an interval of 0.15?m were collected for the entire cell height which approximated 10C12?m. Z-stack analyses and other post-acquisition processing were performed on ZEN Black software (Carl Zeiss). Calcium measurements VSMC were seeded on 3D biochips. Calcium measurements in 3D biochips were performed as previously explained with modifications37. Briefly, cells in 3D biochips were PR-619 serum-starved for 12?h and loaded with 5?M of calcium green (dissolved in DMSO) for 30?min at room heat, with Hanks PR-619 Balanced Salt answer, (HBSS) supplemented with CaCl2, MgCl2 and 10?mM HEPES. Calcium Green was imaged using Zeiss 510 equipped with 40?x Apochromat objective at acquisition frame rate of 4 fps (250?ms acquisition time), and Calcium Green was excited using Argon ion laser 488 at low transmittivity (1%) to prevent photobleaching. Image stacks acquired were then imported into Fiji/ImageJ. Background subtraction was performed on the time stacks by using a rolling ball radius of 50 pixels. Cytoplasm and nuclear regions of interest (ROI) were chosen by performing a maximum intensity projection of the time-stack and specifying a 5?m radius PR-619 circle within the nuclear and cytoplasmic regions. To convert intensity values to Ca2+ concentration, modified Grynkiewicz equation was used, defined as: is the average fluorescence intensity of the ROI after addition of 100?M BAPTA AM, is the average fluorescence intensity of the ROI after addition of 0.100?M A23187. Integrated Ca2+ was calculated using the trapz() function in MATLAB. FRET imaging MLCK-FRET plasmid is usually a kind gift from Dr. James T. Stull (University or college of Texas Southwestern Medical Center). The MLCK-FRET plasmid is usually a calmodulin-binding based sensor, where calmodulin binding sequence is usually flanked with eCFP and eYFP and exhibits decreased FRET upon binding with calmodulin19,38. Cells expressing MLCK-FRET were imaged using Zeiss LSM 880 (Carl Zeiss, Jena, Germany), at 37?C incubator, fixed with Plan-Apochromat 20?x, equipped with 458?nm and 514?nm Argon ion laser lines for excitation of eCFP and eYFP respectively. Incident excitation light was split using an MBS 458?nm/514?nm beam splitter and collected on a 32-spectral array GaAsp detector. The fluorescence emission was collected from 463C520?nm (ECFP), 544C620?nm (FRET channel and eYFP channel). Intensity based ratiometric FRET were obtained using custom-written scripts in ImageJ and MATLAB. Since MLCK-FRET is usually a single-chain construct, decrease in FRET, and increase in MLCK binding to.