A) Upper panel shows semi-quantitative PCR of HoxD10 mRNA in control or HoxD10 transfected T4-2 cells. Lower panel shows corresponding western blot of HoxD10 protein.
B) Upper panel shows phase contrast photomicrographs of control and HoxD10-expressing T4-2 cells on tissue culture plastic for 48 hours. Lower panels show phase contrast photomicrographs, IF co-staining for β4 integrin (red), and merged image with DAPI (blue) in control T4-2 or HoxD10-expressing T4-2 cells grown in 3D lrECM for 72 hours.
C) Real time PCR of VEGF mRNA levels in non-malignant S1 cells, control or HoxD10-expressing T4-2 cells grown in 3D lrECM for 72 hours. (** p<0.05, n=4). Corresponding ELISA showing relative levels of VEGF protein secreted by S1, T4-2 and HoxD10-reverted T4-2 cells cultured on 3D lrECM for 72 hours. (** p<0.05, n=5).
D) Inset: Schematic of co-culture model of HMEC-1 on collagen-coated transwells with either S1, T4-2 or HoxD10-expressing T4-2 cells cultured in 3D lrECM in the lower chambers. Plot shows migration of EC after 4 hours of co-culture with lrECM only (negative control; dots), S1 cells (white), T4-2 cells (black), HoxD10-expressing T4-2 cells (grey), or pretreatment with neutralizing antibody against VEGF (striped). Mean number of cells migrated +/- S.D. from five different fields (20x) for each of 4 independent experiments is shown (** p<0.05).

A) Upper panel shows phase contrast photomicrographs of polarized S1 or S1-EGFR cells cultured in 3D lrECM for 72 hours. Lower panels shows corresponding IF staining for EGFR (red) and merged image with DAPI staining (blue).
B) Left panel shows real-time PCR VEGF mRNA levels in S1 (white) or S1-EGFR cells (horizontal stripes) cultured in 3D lrECM for 72 hours, expressed as percentage of VEGF mRNA levels by malignant T4-2 cells in the same conditions (n=4). Right side shows corresponding EC migration over 4 hour co-culture with either S1 (white) or S1-EGFR cells (horizontal stripes) in 3D lrECM in the lower chamber. Results show the mean of migrated EC from five different fields (20x) for each of three independent experiments (n=4).
C) Upper panel shows western blot of HIF1α in control T4-2 or HoxD10-expressing T4-2 cells cultured in 3D lrECM for 72 hours. Total protein loading is shown by corresponding levels of β-actin. Lower panel shows EMSA with nuclear extracts from control or HoxD10-expressing T4-2 cells incubated with a HIF1α consensus oligonucleotide. Upper arrow shows the position of the protein complexes and lower arrow shows free, unbound probe.

Schematic summarizing the impact of tissue organization on VEGF expression in non-malignant, malignant or malignant breast epithelial cells re-polarized with HoxD10.

A) Unsupervised hierarchical clustering using 22946 probes for ten variations showing distinct transcriptional profiles of S1, T4-2 and T4-2 cells treated with different drugs.
B) Identification of 294 genes significantly associated (P < 0.001) with colony organization in S1, T4-2 and T4-2 cells reverted with different agents. 204 were highly expressed in disorganized (T4-2) colonies and 90 were more highly expressed in organized (S1 and T4-2-reverted) colonies.
C) Profile of seven genes from Fig 1B, belonging to the GO:0001525 class of angiogeneic regulators high in malignant T4-2 and low in polarized S1 and reverted T4-2 cells. The indicated genes correspond to the following Affymetrix probes: VEGF 210512_at, VEGFC 209946_at, CEACAM1 209498_at, TNFRSF12A 218368_s_at, ANGPTL4 221009_s_at, NF1 211094_s_at, ELK3 206127_at.
D) Relative fold-changes detected in angiogenesis-related genes between control (IgG) or EGFR blocking antibody (mAb225) treated T4-2 cells.

A) Upper panel shows western blot of HoxA10 in control, HoxD10 or HoxA10 expressing MDA-MB-231 cells and HeLa cells. Corresponding phase contrast photomicrographs of control, HoxA10, or HoxD10-expressing MDA-MB-231 cells in 3D lrECM for 72 hours and IF staining for β4 integrin. Proliferation is indicated by the % Ki-67 index expressed as the mean +/- SEM (** p<0.05).
B) Real-time PCR of VEGF mRNA in control (black), HoxD10- (gray) and HoxA10- (speckled) expressing MDA-MB-231 cells cultured in 3D lrECM for 72 hours. (** p<0.05, n=4)
C) Migration of EC after 4 hours of co-culture with lrECM only (negative control; dots), control MDA-MB-231 (black), HoxD10- (gray) and HoxA10-expressing MDA-MB-231 cells (speckled), or EC pretreated with a neutralizing antibody against VEGF and co-cultured with control (dark grey stripes) or HoxA10-expressing MDA-MB-231 cells (light grey stripes) all cultured in 3D lrECM. Results show the mean of migrated EC in a total of five different fields (20x) +/-s.d. for each of three independent experiments (** p<0.05).

A) Left panel: levels of HoxD10 in control, HoxD10-expressing or T4-2 cells expressing both HoxD10 and dominant active Rac1. Right panel shows western blot for GTP-loaded Rac 1 (upper) in corresponding cells. Lower right panel shows total protein determined by re-probing with β actin.
B) Upper panel shows phase contrast microscopy of T4-2, HoxD10-expressing T4-2 or T4-2 cells expressing both HoxD10 and Rac1L61 cultured in 3D lrECM for 72 hours. Middle panel shows corresponding IF staining for β4 integrin (red) and lower panel shows merged images for β4 integrin (red) and nuclear DAPI (blue). Ki-67 labeling index was assessed after 72 hours in 3D lrECM; results are expressed as the mean +/- SEM for 10 independent measurements.
C) Real-time PCR showing VEGF mRNA in control T4-2 (black), T4-2 expressing HoxD10 (grey), and T4-2 cells expressing HoxD10 and Rac1L61 (checkered) after 72 hours in 3D lrECM (** p<0.05, n=4). Corresponding ELISA showing VEGF protein secreted by each cell type (n=3, **p<0.05).
D) Relative migration of EC over 4 hours of co-culture with lrECM only (negative control; dots), control T4-2 cells (black), HoxD10-expressing T4-2 cells (grey) and T4-2 cells expressing HoxD10 and Rac1L61 (checkered), cultured for 72 hours in 3D lrECM. Results show the mean of five different fields (20x) for each of three independent experiments (** p<0.05).