Lysophosphatidate Reverses G2/M Arrest by Taxol Figure 8. Differential expression of autotaxin and LPA1-3 receptors in MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells. Results show mRNA concentrations expressed relative to that for cyclophilin for (A) ATX (B) LPA1 (C) LPA2 and (D) LPA3 in cells incubated for 26 h in the presence or absence of Taxol. Results are means 6 SD from three independent experiments for ATX and LPA1 and six independent experiments for LPA2 and LPA3. *P,0.05 and ** p,0.01. doi:10.1371/journal.pone.0020608.g008 Figure 9. Involvement of LPA1/3 and LPA2 receptors in LPA- induced displacement of Taxol from polymerized tubulin in MCF-7 and MDA-MB-231 cells. MCF-7 (Panel A) and MDA-MB-231 cells (Panel B) were preincubated for 26 h with [3H]Taxol as in Fig. 7. After this, 5 mM LPA or 1 mM VPC51299, a LPA1/3 receptor antagonist, was added as indicated and the incubations were continued for a further 12 h. Polymerized tubulin was then isolated and the distribution of [3H]Taxol was determined. Results are means 6 SD for three independent experiments. *P,0.05 comparing cells incubated in the absence of LPA with those incubated with LPA and/or the LPA1/3 antagonist, VPC51299. doi:10.1371/journal.pone.0020608.g009 containing 50 nM [3H]Taxol (6,660 Ci/mol). Cells were incubat- ed for the indicated times and medium was collected. Cells were washed three times with ice-cold RPMI 1640 medium containing 0.1% BSA to remove extracellular Taxol. The cell lysates were then collected by scrapping twice with 0.5 ml of ice-cold methanol. Chloroform (1 ml) was added to the combined methanol extracts followed by 0.9 ml of 2 M KCl. The mixture was shaken and centrifuged. Radioactivity in both cell lysates and media were measured by scintillation counting. Cell extracts were also analyzed on silica gel 60 thin layer chromatography plates (Merck, Darmstadt, Germany) after development with toluene/ acetone/formic acid (60:39:1, by vol.) [37]. The distribution of 3H was determined with a BioScan 200 Imaging Scanner followed by scintillation counting of various areas of the plate. Measurement of Taxol association with polymerized tubulin Incorporation of Taxol in the soluble (cytosolic) and polymer- ized (non-cytosolic) fractions of the cells was determined by a standard method as described previously [59,60]. Briefly, 26106 cells were seeded in 6 cm dishes. After growing cells to confluence, the medium was replaced with 3 ml RPMI 1640 containing 10% 3 of charcoal-treated FBS and 50 nM [ H]Taxol (0.5 mCi). Cells were incubated for 26 h. Then the incubation was continued for 3 additional 12 h with [ H]Taxol in the presence and absence of LPA (5 mM) and different inhibitors as described in the text. Cells were then collected and suspended in 200 ml of hypotonic lysis buffer (1 mM MgCl2, 2 mM EGTA, 0.5% NP-40, 20 mM Tris- HCl at pH 6.8 containing protease and phosphatases inhibitors) and incubated for 5 min. Polymerized tubulin was collected in a pellet by microcentrifugation at 18000 x g for 10 min. The supernatant (200 ml) containing the cytosolic fraction was transferred to another tube. The pellet containing the polymerized fraction was resuspended in 200 ml of hypotonic lysis buffer. Protein concentrations for each fraction were measured using the BCA assay (Thermo Scientific). A sample of each fraction (20 ml) PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e20608