Figure 1.
Actin polymerization increases in response to DNA damage.
A. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and images were captured at the indicated time points. B. The cell length and width were analyzed with Image J software in ≥100 cells per condition. C. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of phalloidin was measured with Image Pro Plus software. Scale bar, 10 µm. D. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular F-actin levels (phalloidin intensity/DAPI intensity). E. Cells were treated with ETO (10 µM) at indicated time points, and then whole cell extracts were analyzed by western blotting using anti-γH2AX antibody (a). U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h. Then, immunofluorescence was performed to detect the signal of γH2AX (b). Scale bar, 10 µm. All Statistical differences were determined by One-way ANOVA. Results are presented as means ± SD of values from three independent experiments. ETO, etoposide.
Figure 2.
Actin polymerization modulates p53 cellular accumulation.
A. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, cells were harvested and the whole cell proteins were extracted for western blotting to measure p53 protein levels. B. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of FITC staining p53 was measured with Image Pro Plus software. Scale bar, 10 µm. C. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular p53 levels (FITC intensity/DAPI intensity). D. U2OS cells were transfected with HA-actin or pcDNA, then treated with ETO (10 µM) or untreated as control at indicated time points. The cells were harvested, RNA extraction and Real Time-PCR were carried out. The mRNA content of p53 was normalized to that of GAPDH and the normal cells’ mRNA level was valued as 1. Data (mean±SD) were from three independent experiments. E. U2OS cells transfected with HA-actin were treated with ETO (10 µM) or untreated as control for 12 h. Whole cell proteins were extracted and western blotting was performed. F. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h to change actin dynamics, then treated with ETO or not. Cells were harvested, and whole cell proteins were extracted for western blotting to detect p53 protein levels in different conditions (a). Results were analyzed with Image J software (b). All Statistical differences were determined by One-way ANOVA. Jas, Jasplakinolide; CD, Cytochalasin D.
Figure 3.
p53 binds to polymeric actin in the cytoplasm.
A. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and followed with ETO (10 µM) treatment for another 12 h. The cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blottings of immunoprecipitated actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b). B. U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h and treated with or without ETO (10 µM) for another 12 h. The cells were then harvested and the whole cell extracts were incubated with GST or recombinant GST-p53. The bound proteins were analyzed by western blotting with anti-actin antibody (top panel). GST and GST–p53 were stained with Coomassie Blue (middle panel). Arrows show the position of GST and GST–p53. Whole cell extracts were immunoblotted with antibody against GAPDH to confirm equal loading (bottom panel) (a). Western blottings of actin were analyzed using Image J software. Results are presented as means ± SD of values from three independent experiments (b).C.U2OS cells were treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h before YFP-p53 transfection. Twenty-four hours after YFP-p53 transfection, cells were treated with ETO (10 µM) for another 12 h or untreated as control and analyzed by confocalmicroscopy (a). Scale bar, 10 µm. The colocalization ratio of F-actin and YFP-p53 of more than 100 cells were calculated using Image J software. Results are presented as means ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. *, P<0.05; **, P<0.01.
Figure 4.
Actin polymerization impairs p53 nuclear import.
A. Cells were transfected with siRNA-cofilin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h before YFP-p53 transfection. 24 h after YFP-p53 transfection, cells were treated with ETO (10 µM) or untreated for another 12 h. Cells were then analyzed by confocalmicroscopy. Scale bar, 10 µm. B. Fluorescence values of more than 100 cells were calculated using Image J software. Results are presented as means ± SD of values from three independent experiments. C. Cells were transfected with control siRNA or cofilin siRNA, whole cell protein extraction and western blotting were then carried out to detect cofilin suppression efficiency. Panels of western blotting were analyzed with Image J software. Results are presented as means ± SD of values from three independent experiments. D. Cells were transfected with siRNA-cofilin or treated with Jas (50 nM) or CD (0.01µg/ml) for 2 h before cells were treated with ETO (10 µM) or untreated as control for another 12 h. Cytoplasmic protein and nuclear proteins were then extracted, and western blotting was performed. Oct-1 (octamer transcription factor 1) was used as nuclear protein marker and GAPDH was used as cytoplasmic protein marker (a and c). Panels of western blotting were analyzed with Image J software. Results are presented as means ± SD of values from three independent experiments (b and d). All Statistical differences were determined by One-way ANOVA. *, P<0.05; **, P<0.01.
Figure 5.
Actin polymerization affects the Ser315 phosphorylation and ubiquitination of p53.
A. U2OS cells transfected with HA-actin were treated with ETO (10 µM) or untreated as control for 12 h. The cells were then harvested and whole cell proteins were extracted for western blottings with indicated antibodies. B. Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments. C. U2OS cells transfected with HA-actin. At 24 h after the transfection, cells were treated with or without ETO for 12 h, followed by the treatment of 20 µM MG132 for 6 h. Whole cell extracts were immunoprecipitated with anti-p53 (DO-1) antibody and analyzed by western blotting with anti-p53 (FL-393) antibody. D. Cells were transfected with Aurora kinase A siRNA, and 24 h after transfection, cells were treated with ETO (10 µM) or untreated as control for 12 h. Then, cells were harvested and whole cell proteins were extracted for western blotting (a). Western blottings were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). E. Cells were harvested and subjected to immunoprecipitations using anti-Aurora A antibody and analyzed by western blotting. F. Cells were transfected with actin or actin siRNA, or treated with ETO (10 µM, 12 hours) or untreated as control, or CD (0.01µg/ml) and Jas (50 nM) for 2 h. Cells were then harvested and subjected to immunoprecipitations using anti-p53 antibody and analyzed by western blotting (a). Western blotting results were analyzed using Image J software. Results are presented as mean ± SD of values from three independent experiments (b). All Statistical differences were determined by One-way ANOVA. **, P<0.01.
Figure 6.
The impact of actin polymerization on p53 leads to the alteration of p21 expression.
A. Cells were transfected with HA-actin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h. Then, cells were treated with ETO (10 µM) or untreated as control for 12 h. Whole cell proteins were extracted and western blotting was performed to measure p21 protein levels. B. Western blotting were analyzed with Image J software, and the results are presented as mean ± SD of values from three independent experiments. C. Cells were transfected with HA-actin or treated with Jas (50 nM) or CD (0.01 µg/ml) for 2 h. Then, cells were treated with ETO (10 µM) or untreated as control for 12 h. Real-Time PCR was performed, mRNA content of p21 was normalized to that of GAPDH and the normal cells’ mRNA level was valued as 1. Data (mean±SD) were from three independent experiments. All Statistical differences were determined by One-way ANOVA. **, P<0.01.