Cells were grown on glass-bottom plates to enable live microscopy imaging. After incubation for 20-hour without or with 1μM rapamycin, cells were loaded with 5 μM Fura2-AM (TEFLabs, Austin, TX) at 37°C for 30 min. Cells were then washed with DPBS (Dulbecco's Phosphate-Buffered Saline) and observed under a 40× objective lens with a Nikon Eclipse Ti-E microscope controlled by Elements software. Cytosolic calcium was observed by recording calcium-bound Fura excitation fluorescence at 340/380 nm and emission at 510 nm. Baseline calcium was observed for 2 minutes prior to data acquisition. Fluid-shear stress was then applied to cells utilizing an Instech P720 peristaltic pump with an inlet and outlet setup. The fluid was perfused on the glass-bottom plates at shear stress of 0.2, 0.6 or 1.0 dyne/cm2 for epithelial cells and 2.0, 5.0 or 8.0 dyne/cm2 for endothelial cells. After each experiment, the maximum calcium signal was obtained by perfusion of ATP (10μM) to confirm cell viability. Conditions for all experiments were maintained at 37°C and 5% CO2 in a stage top cage incubator (okoLab, Burlingame, CA). Calcium analysis was then followed a standard calculation as previously described (Upadhyay et al., 2014 (link)).