Nanoscope software

Single dissociated cells were put on a glass bottom dish in 1×MBS-H. They were compressed using a Bioscope Atomic force microscope (AFM) (Veeco) mounted on an inverted optical microscope (Axiovert S100 TV; Zeiss) with a Silicon Nitride cantilever with a nominal spring constant of K = 0.01 (MLCT; Veeco) mounted with a 10 µm polystyrene bead (Polysciences). Force-distance (FD) curves were obtained with the Nanoscope software (Digital Instruments). FD curves were plotted as the deflection of the cantilever as it was lowered in the z-axis to make contact with, and compress single cells. The elastic modulus was calculated by fitting the first 200 nm of each FD curve using a Hertzian compression model for a spherical indenter compressing a spherical substrate. Code kindly provided by X.Y. Chau and P. Grütter, McGill University, available at http://spm.physics.mcgill.ca/research-projects/afm-in-fluids/mechanical-properties-of-neurons. Multiple approaches were taken for each cell and the average elastic modulus for each cell was used as a single data point. Statistical analysis was done using a one-way ANOVA followed by Tukey’s HSD post hoc test for individual comparisons.

Sample preparation for atomic force microscopy (AFM) was performed at RT. Typically, 3–6 μl of different ASN species diluted in corresponding buffers to a working concentration of 1 μM were applied to a freshly cleaved muscovite mica substrate (Ted Pella, Redding, CA) and incubated for 1 min. The mica surface was then rinsed with 7–200 μl of double-processed tissue culture water (Sigma-Aldrich, St. Louis, MO, USA) to remove salts and loosely bound proteins. AFM images were recorded on a MultiMode^TM SPM (Digital Instruments, Santa Barbara, CA) equipped with an E-Scanner using etched silicon NanoProbes (model RTESP; Veeco Instruments, Mannheim, Germany). All measurements were performed in the tapping mode with scan rates of ~ 0.5 Hz. Images were processed using NanoScope software (Digital Instruments).