Chondramide does not influence EGF-R signalling.

<p>MDA-MB-231 cells were treated with Chondramide (1 or 24 h), stimulated with EGF (5 min) and analyzed via Western blot analysis on EGFR, Akt and Erk. Left panel: one representative Western blot is shown. Right: Densitometric analysis of Western blots. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3.</p

Chondramide diminishes metastasis <i>in vivo</i>.

<p>1×10⁵ 4T1-Luc cells were injected intravenously into pretreated (0.5 mg/kg ChB) and untreated BALB/cByJRj mice. (A) 8 days after cell inoculation mice were sacrificed, lungs were harvested and used for recording bioluminescence signals. Each lung was imaged from the dorsal and ventral side. Color bar scales were equalized. (B) Quantitative evaluation of metastasis to the lungs. Region of interest were defined (ROI) and total luciferin signal in ROIs was calculated as photons/second/cm² (total flux/area). Ten lungs per group, *, p<0.05 (t-test, unpaired). (C) Mouse weight over treatment period. Weight of treated (ChB) and untreated (DMSO) mice from day of cell inoculation (day 0) to euthanasia (day8) is shown for each group.</p

Chondramide diminishes contractility.

<p>(A) MDA-MB-231 cells were pretreated for 24 h as indicated, embedded in matrigel containing fluorescent beads and pictures were taken over 4 h every 15 min. Cellular, contractile force on the surrounding matrix was visualized via bead movement towards the cell and analyzed using PIV analysis. Upper panel: Representative images of cells (red) and fluorescent beads (green) t = 0. Lower panel: PIV analysis of bead velocity in the 2D projection. Color codes show bead velocity indicating applied force. Direction of vectors indicates averaged direction of bead movement. (B) For quantitative analysis the radial velocity (bead velocity towards the cell center) per data point was calculated. Minimum 13 cells per condition were analyzed. *, p<0.05 One-way ANOVA, Tukey post-test. (C) Phosphorylation of the Rho-GEF Vav2 was tested via Western blot analysis upon EGF stimulation (n = 3); * p<0.05 vs. control, One-way ANOVA, Tukey post-test.</p

Chondramide affects activation of the RhoGTPase Rho.

<p>(A) A Rac1 pull down was performed for untreated and Chondramide treated cells (24 h) upon EGF-stimulation (5 min). (B) After same treatment a Rho pull down was conducted. (C) Myosin light chain 2 (MLC2) was analyzed on its activation state via Western blot analysis upon EGF stimulation (5 min). A,B,C: Left panel: one representative Western blot is shown. Right panel: Densitometric analysis of Western blots. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3.</p

Treatment with Chondramide reduces breast cancer cell migration, invasion and adhesion.

<p>(A) Chondramide treated and untreated MDA-MB-231 cells were allowed to migrate in a Boyden chamber for 16 h. (B) Chondramide inhibits invasion of MDA-MB-231 cells through matrigel in Boyden chamber (48 h). A,B: For positive control (PC) lower compartment was filled with medium plus 10% FCS, for negative control (NC) only medium without FCS was added. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3. (C) Pretreated MDA-MB-231 cells were seeded freshly on indicated surfaces and counted after fixation. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3. (D) Cells from C were stained for F-actin, nuclei and vinculin. Bar represents 50 µm.</p