Fig. 3

Nucleus spreading and cytoplasmic CFL2/F-actin reorganization under steady shear flow of 1.1 Pa for 24 h. DNA was visualized with Hoechst 33342 (a) for quantifying cell density (b), cell or nucleus area (c), as well as nucleus/cell area ratio (d). Nucleus stiffness was measured using AFM assay and presented by their Young’s moduli (e). Simple western immunoblots (f) showed CFL2 proteins extracted from the whole cell, cytoplasm, or nucleus under static and shear conditions. Here ACTB served as the loading control for total and cytoplasmic proteins and LMNB1 as the loading control for nuclear proteins. CFL2 expressions in whole cells (g), cytoplasm (h), and nucleus (i) were validated with immunoblots. Data are presented as the mean ± SE of normalized chemiluminescence in three replicates. Cytoskeletons (j) were imaged to quantify CFL2 to F-actin ratio (k) and intensity correlation quantile (ICQ) of F-actin and CFL2 (L). Open arrows in j indicated CFL2 aggregation close to nuclear periphery. The numbers of measured colonies and positions in each colony (m, n) are (5, 8) in e. The numbers of tested replicates and measured colonies, (M, m), are (4, 32) for static control, (3, 36) for fluid shear, and (3, 30) for TSA treatment in a–d, as well as (3, 24) for static control and (3, 45) for fluid shear in k and l. Flow direction was indicated by solid arrows at the bottom. Bar = 50 μm in a and 20 μm in j