Figure 1.
Cucurbitacin B (CuB) induced VASP phosphorylation and clustering in A375 cells.
(A,B) Western blot analysis showing the dose- (A) and time- (B) dependent effect of CuB on VASP phosphorylation. (C) Immunofluorescence microscopy analysis showing the co-localization (yellow) of VASP (red) and actin (green) with nuclei (blue) counter staining after 0.1 μM CuB treatment. (D) Co-localization analysis of VASP and actin of C. Both PDM images and ICQ values (right panel) are shown. (E) CuB induced cofilin-actin rod formation. Cells were immunostained with anti-β-actin (green) and anti-cofilin (red) antibodies with nuclei (blue) counter staining. (F) Influence of cofilin knockdown on CuB-induced VASP activation. (G) Influence of cofilin knockdown on actin distribution. Magnified images of the boxed areas (merged images) are presented in C, E and G. Scale bars: 10 μm (5 μm in magnified images).
Figure 2.
VASP knockdown mitigated CuB (0.1 μM)-induced actin aggregation, cofilin-actin rod formation and cell deformation.
(A) Knockdown of VASP did not affect CuB-induced cofilin dephosphorylation as revealed by western blot analysis. (B) Effect of VASP knockdown on CuB-induced cell deformation as observed using phase-contrast microscopy. Scale bars: 20 μm. (C) Immunofluorescence microscopy analysis of the influence of VASP (red) knockdown on CuB-induced actin (green) aggregation. Scale bars: 10 μm (5 μm in magnified images).
Figure 3.
H89, but not MDL12330A (MDL), pretreatment suppressed CuB-induced VASP activation, actin aggregation and cell deformation.
(A) Western blotting showing the effects of H89 and MDL pretreatment on CuB-induced VASP activation and cofilin dephosphorylation. (B,C) CuB decreased the levels of cAMP in a time- and dose-dependent manner (B). H89 and MDL pretreatment affected the levels of cAMP in CuB-treated cells (C). The cAMP level of forskolin (10 μM)-treated cells was set as 100% activity (B,C). (D) Immunofluorescence analysis of the influence of H89 pretreatment on the distribution of VASP (red) and actin (green) in CuB-treated cells. Scale bars: 10 μm (5 μm in magnified images). (E) Co-localization analysis of VASP and actin of D. (F) Effect of H89 pretreatment and MDL pretreatment on CuB-induced cell deformation as observed using phase-contrast microscopy. Magnified images of the boxed areas are presented on the right. Scale bars: 20 μm.
Figure 4.
Gα13 and RhoA were involved in CuB-induced VASP activation and actin cytoskeleton disruption.
(A) The expression of Gα13 and RhoA was not affected after CuB treatment as evidenced by western blotting. (B,C) Western blot analysis showing the effect of RhoA (B) or Gα13 (C) knockdown on CuB-induced VASP activation and cofilin dephosphorylation (B,C) and JNK activation (C). (D,E)Immunofluorescence microscopy analysis showing the distribution of RhoA (green) and actin (red) in of RhoA-knockdown cells (D) and the distribution of VASP (red) and actin (green) in Gα13-knockdown cells (E). Scale bars: 10 μm (5 μm in magnified images). The white arrowheads indicate the cell in which Gα13 was knocked down (E). (F) Effect of Gα13 and RhoA knockdown on CuB-induced cell deformation observed using phase-contrast microscopy. Scale bars: 20 μm.
Figure 5.
A proposed mechanism depicts CuB-induced actin aggregation and VASP activation via the Gα13/RhoA/PKA pathway.
Dashed arrows and question marks indicate unidentifed/unconfirmed signaling components.