Lysophosphatidate Reverses G2/M Arrest by Taxol Figure 7. LPA causes dissociation Taxol from the polymerized tubulin compartment. Approximately 26106 cells were seeded in 6 cm dishes. After growing cells to confluence, the medium was replaced with 3 ml RPMI 1640 containing 10% of charcoal-treated FBS containing 50 nM [3H]Taxol (0.5 mCi). Cells were preincubated for 26 h. After this, 5 mM LPA and different concentrations of inhibitors were added as indicated. Polymerized tubulin (Panel A) and soluble fractions (Panel B) were then separated and the distribution of [3H]Taxol was determined. Panels C and D show the dose response curves for LY294002 and Wortmannin, respectively. Results are means 6 SD for three independent experiments. Panels A and B, * a significant difference of P,0.05. doi:10.1371/journal.pone.0020608.g007 attention. No present cancer treatment depends on the inhibition of ATX or LPA signaling. This work emphasizes the importance of developing therapeutic agents that decrease ATX activity and signaling through LPA receptors. These agents could provide valuable adjuvants to increase the efficacy chemotherapy and surgery. Materials and Methods The sources of most of the materials and details of methods including the preparation of delipidated calf serum have been described [7]. Cells Characterized MCF-7, MDA-MB-231 and MDA-MB-468 cells were obtained from the American Type Culture Collection (Manassas, VA) and they were used within six months after resuscitation. Materials Monoclonal anti-a-tubulin, sodium pyruvate, NADH, RNAase A, goat and donkey serum, sodium borate, and propidium iodide were from Sigma-Aldrich (Oakville, ON, Canada). Bromodeoxy- uridine (BrdU), mouse anti-BrdU and FITC goat anti–mouse IgG antibody were purchased from BD Pharmingen (Mississauga, ON, Canada). [o-benzamido-3H]Taxol was obtained from Moravek Biochemicals (Brea, CA, USA). Anti-phospho-histone H3 was from Millipore (Billerica, MA, USA). We purchased TBB (casein kinase-2 inhibitor) from Tocris Biosciences (Ellisville, MI, USA). Cell culture and bromodeoxyuridine/propidium iodine double staining Bromodeoxyuridine/propidium iodine double staining was performed as described previously [57]. For nuclei isolation, we optimized incubation times and temperatures for enzymatic digestion in MCF-7 cells (5 ml of 0.04% pepsin in 0.1 N HCl for 20 min at room temperature). Samples were analyzed with a Calibur Flow Cytometer (Becton Dickinson, San Jose´, CA, USA) using Cell Quest software. Immunocytochemistry Flame-sterilized glass coverslips (18 mm) were placed in 12-well culture plates and about 26105 MCF-7 breast cancer cells were plated and grown to confluence. Cells were stained with DAPI, monoclonal anti-a-tubulin and polyclonal anti-histone H3.1 (Phospho-Ser 10) [58]. Measurement of Taxol uptake, distribution and metabolism After growing cells to confluence, the medium was replaced with 1.5 ml RPMI 1640 containing 10% of charcoal-treated FBS PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e20608