Podoplanin mRNA transcription and transcriptional initiation in the human osteoblast-like cell line MG63. A) PDPN transcription was analyzed using primers specific for podoplanin cDNA (see "Material and Methods"). The human cell lines are: MG63 and Saos-2, osteosarcoma cells; HUVEC, umbilical vein endothelial cells; EAhy926, endothelial-like cells; Met5A, pleural mesothelial cells; A549, type II alveolar lung carcinoma cells; Raji, Burkitt's B-lymphoma cells; JY, B-lymphoblastoid cells; K562, erythroleukemia cells; CCL, "centrocyte-like" B-lymphocytes; HL60, promyeloid cells. B) Real-time PCR analysis of PDPN transcription in MG63 versus Saos-2 cells. Results ± S.D. of three independent PCRs are shown. Transcript levels are relative to GAPDH mRNA content. C) Northern blot analysis of podoplanin mRNA fragments in MG63 versus Saos-2 cells. D) RT-PCR determination of transcription initiation sites in MG63 cells. The lane numbers correspond to the RT-PCR 5' primers in Table 1. They were tested together with 3' primer hPPrev (+114/+96) for their ability to amplify the corresponding sequences in cDNA preparations of MG63 cells. PCR products are shown on an agarose gel stained with ethidium bromide. Lanes L: DNA ladder, Lane C: Negative control containing no template DNA for PCR reaction. E) Sequencing results of 48 clones retrieved by 5' RACE from MG63 cells. The 5' cDNA endings and their frequency are indicated above the sequence stretch.