A. Immunohistochemical analysis of normal, primary breast cancer tumors and metastatic tumors: comparison of the palladin staining in the tissue sections demonstrates an apparent increase in staining in metastatic tumors. B. Palladin levels are increased in primary tumors and metastatic tumors: western blots were used to determine the levels of palladin in normal, primary breast cancer tumors and metastatic tumors. GADPH (a standard house-keeping protein) was used as a loading control.

A- MCF7, MDA-MB-231 and SUM159 cells were plated on FBS-coated coverslips and treated with the phorbol ester PDBu for 20 minutes. Co-labeling with rhodamine-phalloidin and polyclonal anti-cortactin antibody was used to detect podosomes. Note that only the invasive MDA-MB-231 and SUM159 breast cancer cell lines form podosomes after PDBu treatment and not the non-invasive MCF7 cell line. B- MDA-MB-231 and SUM159 cells were plated on FBS-coated coverslips and treated with the phorbol ester PDBu for 20 minutes. After fixation, endogenous palladin was detected by immunofluorescence. Co-labeling with phalloidin and anti-palladin antibody reveals that palladin was consistently detected in podosomes of the both invasive cell lines. Top two panels: Low magnification images of MDA-MB-231 and SUM159 breast cancer cell lines. Bottom: High magnification image of SUM159 cells to show detail. C- SUM159 cells transfected with control siRNA oligos and siRNA oligos targeting palladin were plated on FBS-coated coverslips overnight and then treated with PDBu for 20 minutes. Left panel- Western blot analysis was used to detect palladin levels in cells treated with control oligos (C) or palladin- specific siRNA oligos (KD). RNA interference reduced expression by ∼90%. Right panel- Podosome formation was significantly reduced in palladin knockdown cell (KD, 19 ± 5 %), as compared with control cells (C, 36 ± 3 %). Results are representative of three independent experiments in which at least 300 transfected cells were counted. Scale Bar = 10μm

A- Western blot analysis shows that treatment with adenovirus vector containing myc-palladin in MCF7 cells leads to palladin levels comparable with those found in invasive cell lines such as MDA-MB-231 and SUM159. B- GFP and palladin-overexpressing MCF7 cells were plated on FBS-coated coverslips and treated with the phorbol ester PDBu for 20 minutes. After fixation, palladin was detected by immunofluorescence. Co-labeling with phalloidin and anti-myc antibody reveals that myc-palladin was consistently detected in podosomes of infected cell lines. C- Podosome formation was induced in palladin overexpressing cells (OE, 10 ± 2%) as compared with control cells (C, 0 %). Results are representative of three independent experiments in which at least 100 transfected cells were counted. Scale Bar = 10μm

A- Western blot analysis was used to determine the amount of palladin protein in whole-cell lysates of eight different human breast cancer cell lines: T47D, BT474, ZR75.1, MCF7, BT549, Hs578T, MDA-MB-231 and SUM159. B- Quantification of palladin protein levels in breast cancer cells. The ratio of palladin to total cellular tubulin is shown on the left axis, and this amount was found to correlate closely with the invasive potential of the eight cell lines.

A- Western blot analysis shows that treatment with palladin siRNA leads to a ∼90% reduction in palladin expression in SUM 159 cells (KD), as compared to cells treated with control siRNA (C). B- Transwell migration assay of control siRNA (C) and palladin siRNA (KD) transfected MCF7 and SUM159 cells. Palladin knockdown reduces the number of cells migrating through a transwell filter by ∼60% in SUM-159 cells, while the non-invasive MCF-7 cells are non-migratory either with or without palladin siRNA treatment. C- Matrigel invasion assay of control siRNA (C) and palladin siRNA (KD) transfected MCF7 and SUM159 cells. Palladin knockdown reduces the number of cells invading through a Matrigel-coated transwell filter by ∼50% in SUM-159 cells, while the non-invasive MCF-7 cells are non-invasive either with or without palladin siRNA treatment. For both assays, cells on the lower surface of the filter were fixed and counted 20 hours after plating (Representative microphotographs of migrating and invading cells are included).

A- The levels of palladin in MCF7 and SUM159 cells infected with adenovirus expressing either GFP (C, control) or myc-palladin (OE, overexpression) were assessed by Western blot. B- Transwell migration assay of MCF7 (left) and SUM159 (right) expressing either GFP (C) or myc-palladin (OE). C- Matrigel invasion assay of control MCF7 (left) and SUM159 (right) expressing either GFP © or myc-palladin (OE). For both assays, cells on the lower surface of the filter were fixed and counted 20 hours after plating (Representative microphotographs of migrating and invading cells from three different experiments are included).