Quantifying Nuclear Stiffness by AFM

SMCs in 35 mm glass coverslip-bottom dishes (World Precision Instruments) were incubated with Dox for 24 hours to induce nuclear lamin expression. Nuclear stiffness (Young’s modulus) in adherent cells was measured on a NanoWizard 4a Bioscience AFM (JPK Instruments) coupled with a Zeiss LSM5 confocal fluorescence microscope (53 (link), 54 (link)). A spherical AFM tip with a radius of 500 nm and 0.2 N/m spring constant cantilever (Nanotools, B500-CONT) was placed on top of the cell over the nucleus, identified by staining DNA with Hoechst 33342. Three different areas of the nucleus were sampled with a 2-nN maximum set point. The 3 measurements were used to calculate the average Young’s modulus for a single cell. All measurements were performed at 37° C in HEPES-buffered culture medium. The Young’s modulus was measured in 25 randomly selected cells for each group, and the force curves analyzed with JPK Data Processing software (JPK Instruments). The Young’s modulus was calculated with the Hertz model for a spherical tip and applied to fit the slopes of the approach curve.