Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression

Figure 4

Doxorubicin treatment induces protrusion formation in cells cultured on soft substrates. (a, d) Cells cultured on substrates with elasticities of 2 kPa (a, d) and 30 kPa (d) were treated with or without DOXO (1 μg/mL) for 16 h. (a) Phase contrast images of the cells were obtained with an inverted microscope. Scale bar, 100 μm. (b) The aspect ratio of spheroids cultured on 2 kPa was calculated from the major length to the minor length of an ellipse that fitted each spheroid using ImageJ software. Each bar represents the mean ± SD; . Asterisks, . (c) The number of thin protrusions (20 μm) along the periphery of a cell cluster was quantified using ImageJ software. Data indicate the number of protrusions per 100 μm of the periphery of a cell cluster. Each bar represents the mean ± SD; . Asterisks, . (d) The cells were transfected with the Lifeact-GFP expression vector to label F-actin together with the HA-tagged p53 expression vector before treatment with DOXO. Confocal images of F-actin (green), HA (magenta), and nuclei (DAPI; blue) are shown. Z-stack images with an interval of 1.0 μm were obtained using a confocal microscope, and projected images are shown. Magnified images of F-actin in the boxed regions are also shown. Scale bar, 20 μm. The white arrowheads indicate filopodia-like protrusions (10 μm). (e) The relative number of cells, which have more than three filopodia-like protrusions (10 μm), to total cell number (), is shown.

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