Table of Contents
Journal of Signal Transduction
Volume 2011, Article ID 541851, 13 pages
Research Article

Individual Rac GTPases Mediate Aspects of Prostate Cancer Cell and Bone Marrow Endothelial Cell Interactions

1The Laboratory for Cytoskeletal Physiology, Department of Biological Science, University of Delaware, Newark, DE 19716, USA
2The Center for Translational Cancer Research, University of Delaware, Newark, DE 19716, USA
3Biology Department, Lincoln University, Chester County, PA 19352, USA
4The Delaware Biotechnology Institute, University of Delaware, 320 Wolf Hall, Newark, DE 19716, USA

Received 1 November 2010; Revised 21 February 2011; Accepted 13 April 2011

Academic Editor: Adrienne D. Cox

Copyright © 2011 Moumita Chatterjee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplemental Figure 1 is semi-quantitative PCR analysis for expression of Rac1, Rac3 and RhoG GTPases in the PC-3 prostate cancer cell line. Primers for each PCR are listed in Supplemental Table 1. Represented is a virtual gel produced by semi-quantitative PCR on an Evocycler using FastStart SYBR Green. Each sample is normalized to GAPDH expression from the corresponding sample giving relative band intensities corresponding to expression levels.

Shown in supplemental Figure 2 is the effect of Rac GTPases in the C4-2 LNCaP series prostate cancer cell line. Panel a. Rac isoform expression in C4-2 cells. C4-2 mRNA was harvested and SYBR green-based qPCR performed using primers specified in Supplemental Table 1. Relative expression levels were normalized to GAPDH expression from the corresponding sample and expressed as arbitrary units (a.u.). Panel b. are results of a diapedesis assay after depleting Rac isoforms in C4-2 cells. BMECs were layered onto a Matrigel coated filter and allowed to form a monolayer, 0.5 ml of a suspension of 3.75 x 105 C4-2 cells/ml were added to the BMECs and allowed to undergo diapedesis for 24 h. Compared with untransfected or scrambled controls, depletion of Rac1 or RhoG led to a significant decrease in diapedesis while depletion of Rac3 led to a significant increase in diapedesis. Panel c. C4-2 cells were treated with 100 ng/ml CCL2 in a diapedesis assay as described. Control untransfected (UT) and siRNA control (siScr) cells were compared with untreated/untransfected (UN/UT) C4-2 cells. Cells ability to under go CCL-2 stimulated diapedesis after depletion of Rac1 and RhoG or inhibition of total Rac with iRac was compared to UT and siScr. Rescue experiments were performed by introduction of a siRNA-resistant RhoG led to a significant reversion of RhoG inhibition of diapedesis. For both panels b and c (*) signifies a significant difference between siRNA transfected cells and stimulated controls while (^) signifies a significant difference between siRNA transfected and rescued cells.

Finally, supplemental Figure 3 is a comparison of elasticity in PC-3 and BMECs. PC-3 cells transfected with either a control (siScr) or Rac1-specific SMARTpool siRNA(2) were grown in a monolayer. BMECs were layered onto the PC-3 monolayer, allowed to attach for 30 min and then the elasticity determined by AFM. BMECs in contact with control PC-3 cells had a significant increase in elasticity (*P<.001). Represented are means ± S.D.

  1. Supplementary Figure 1
  2. Supplementary Figure 2
  3. Supplementary Figure 3
  4. Supplementary Table 1