AFM data reveal only a slight increase in TNF-α-activated HUVEC stiffness with substrate stiffness. (A) AFM was used to obtain deflection images for HUVEC monolayers on 0.87-, 5-, and 280-kPa substrates, both under control and TNF-α-treated conditions. Deflection images are 90 μm × 90 μm. (B) AFM was also used to quantify the Young modulus (“stiffness”) of HUVEC monolayers as a function of substrate stiffness in the control (no TNF-α) and after TNF-α treatment. The stiffness of the “cell body” region (raised portion of the cell) and periphery (flattened region just around the raised portion) were quantified separately. Bars represent average stiffness from N force curves from 3 independent experiments. Error bars represent SE. N = 47, 50, and 89 on control monolayers on 0.87, 5, and 280 kPa, respectively, at the cell body. N = 381, 351, and 334 on control monolayers on 0.87, 5, and 280 kPa, respectively, at the periphery. N = 96, 121, and 160 on TNF-α-activated monolayers on 0.87, 5, and 280 kPa, respectively, at the cell body. N = 396, 357, and 399 on TNF-α-activated monolayers on 0.87, 5, and 280 kPa, respectively, at the periphery. ***P < .001 (using ANOVA). On 280 kPa, P < .001 between control and TNF-α at both cell body and periphery using Student t test.