Figure 5. Expression of Net1 with alanine substitutions of the Cdk1 phosphorylation sites causes increased cortical F-actin accumulation in prometaphase cells.

(A) Transfected HA-Net1 responds similarly to Cdk inhibition as endogenous Net1. HeLa cells were transfected with wild type HA-Net1 and treated with vehicle or roscovitin (10 μM, 2 hrs). The cells were then fixed and stained for HA-Net1 (green), DNA (blue), and F-actin (magenta), and imaged by confocal microscopy. Shown are representative z-plane images. Scale bar = 10 μm. (B) Quantification of HA-Net1 plasma membrane localization. Each point represents one cell. Data are aggregated from 3 independent experiments. Bars are median values. ** = p<0.01. (C) Quantification of cortical F-actin staining. Each point represents one cell. Data are aggregated from 3 independent experiments. Bars are median values. * = p<0.05. (D) Net1 SST/AAA expression increases cortical F-actin staining. HeLa cells were transfected with expression vectors for Myc epitope tagged β-Galactosidase, HA-wild type Net1, or HA-Net1 SST/AAA. The cells were then fixed and stained for HA-Net1 or Myc-β-Galactosidase (green), DNA (blue), and F-actin (magenta), and imaged by confocal microscopy. Shown are representative z-plane images. Scale bar = 10 μm. (E) Quantification of plasma membrane association of expressed proteins. Each point represents one cell. Data are aggregated from 3 independent experiments. Bars are median values. ** = p<0.01. (C) Quantification of F-actin at the plasma membrane. Each point represents one cell. Data are aggregated from 3 independent experiments. Bars are median values. *** = p<0.001; n.s. = not significant. (F) Quantification of cortical F-actin staining. Each point represents one cell. Data are aggregated from 3 independent experiments. Bars are median values. * = p<0.05; n.s. = not significant.