Figure 1.
Jasplakinolide induces actin polymerization.
A. HeLa cells were treated with different doses of jasplakinolide (Jasp). G-actin (G) and F-actin (F) fractions were separated as described in Materials and Methods. Equal amounts of G-actin and F-actin fractions were loaded onto SDS-PAGE gels and immunoblotting was done using the actin antibody. Representative data from three independent experiments is shown. B. Changes in the ratio of G-actin and F-actin from immune-blots were quantified using ImageJ. Data are from three independent experiments.
Figure 2.
Actin polymerization lead to YAP de-phosphorylation and nuclear localization.
A. The levels of pYAP are decreased after treatment with jasplakinolide. HeLa cells were treated with 0.5 µM jasplakinolide as described in Materials and Methods. Representative data from three to five independent experiments is shown. B. Changes in the ratio of pYAP/tYAP after jasplakinolide treatment. Densitometric analysis of immunoblotting analyses was done using ImageJ. Error bars represents mean ± SEM, n = 3. *P<0.05. C. Nuclear localization of YAP after Jasplakinolide treatment. HeLa cells were treated with 0.5 µM jasplakinolide and the cells were stained with the YAP antibody as described in Materials and Methods. D. Quantification of number of cells with nuclear YAP signals after jasplakinolide treatment at indicated time points.
Figure 3.
Treatment with jasplakinolide increases expression of CCN growth factors in HeLa cells.
A. Transcript levels for CCN growth factors increased after jasplakinolide treatment. HeLa cells were treated with 0.5 µM jasplakinolide for 10 min. and then cultured for indicated times periods. Representative data from two independent experiments is shown. Error bars represents mean ± SEM, n = 3. *P<0.05, **P<0.001 and ***P<0.0005. B. CYR61 and CTGF protein levels after jasplakinolide treatment. HeLa cells were treated with 0.5 µM jasplakinolide for 10 min. and then cultured for indicated time periods. Protein levels for CYP61 and CTGF were measured from both medium and cells as described in Materials and Method. C. Quantitative estimation of CYR61 and CTGF antigens from both cells and medium fractions. For CYR61, the values of medium fractions are plotted on right axis.
Figure 4.
Knockdown of YAP suppresses CCN growth factor expression after actin polymerization.
CYR61 and CTGF protein levels after YAP knockdown. HeLa cells were transfected with control and YAP siRNAs for 48-PAGE analyses. Immunoblotting was performed using specific antibodies.