Platelet-derived lysophosphatidic acid supports the progression of osteolytic bone metastases in breast cancer
J. Clin. Invest. Ahmed Boucharaba, et al. 114:1714 doi:10.1172/JCI22123 [Go to this article.]

Figure 2
Characterization of MDA-BO2 clones stably transfected to conditionally overexpress HA-LPA₁. (A) Cells transfected with the bidirectional expression vector pBiL-HA-LPA₁ were plated with (+) or without (–) doxycycline (Dox). Two stable clones (nos. 3 and 79) were selected using luciferase activity measurement as an end point. Data are expressed in relative light units (rlu). ^#P < 0.0001 for cells without doxycycline versus cells with doxycycline. (B) Detection of HA-LPA₁ cell surface expression in parental MDA-BO2 cells and in clones no. 3 and no. 79 by flow cytometry using the anti-HA monoclonal antibody. Black and white histograms refer to cells treated without and with doxycycline, respectively. The y axis depicts the number of cells per channel (events), and the x axis depicts the relative fluorescence intensity in arbitrary units (log scale). (C) LPA receptor mRNA expression in parental MDA-BO2 cells and in clones no. 3 and no. 79. Cells were cultured in the absence or presence of doxycycline before total RNA preparation. RT-PCR fragments were separated on a 2% agarose gel and then stained with ethidium bromide. Numbers below the top panel correspond to real-time PCR quantification data of the LPA₁ mRNA copy number for each clone compared with that of the parental MDA-BO2 cells cultured in the absence of doxycycline (mean ± SD; *P < 0.001). No variation of mRNA expression was detected for LPA₂, LPA₃, or GAPDH in the presence or absence of doxycycline.