The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice by Hitendra Singh Chand, Xin Du, Duan Ma, Hector David Inzunza, Shintaro Kamei, Donald Foster, Steven Brodie, and Walter Kisiel Blood Volume 103(3):1069-1077 February 1, 2004 ©2004 by American Society of Hematology

Expression of either wild-type or R24Q human TFPI-2 by stably transfected HT-1080 cells in the presence and absence of G418. Expression of either wild-type or R24Q human TFPI-2 by stably transfected HT-1080 cells in the presence and absence of G418. Stably transfected HT-1080 cell lines, with an initial expression level of approximately 55 ng TFPI-2/mL /day/106 cells, was continuously cultured with passaging in the presence (□, ▵) and absence (▪, ▴) of 0.6 mg/mL G418. The supernatants were assayed weekly for either TFPI-2 (□, ▪) or R24Q TFPI-2 (▵, ▴) expression by a sandwich ELISA as described in “Materials and methods.” Hitendra Singh Chand et al. Blood 2004;103:1069-1077 ©2004 by American Society of Hematology

Growth rates of MT-1080, WT-1080, and QT-1080 cells in culture. Growth rates of MT-1080, WT-1080, and QT-1080 cells in culture. MT-1080 cells, WT-1080 cells, and QT-1080 cells were initially plated at a density of 1 × 105 cells/well in a 6-well plate. Every 7 days, the cells were trypsinized, counted, and replated at the same seeding density. Hitendra Singh Chand et al. Blood 2004;103:1069-1077 ©2004 by American Society of Hematology

Histologic and immunohistochemical analyses of paraffin-embedded subcutaneous and lung tumors. Histologic and immunohistochemical analyses of paraffin-embedded subcutaneous and lung tumors. Tumor sections (5 μ) were either stained with hematoxylin and eosin (H&E) or treated with antibody as described in “Materials and methods.” (A) H&E-stained subcutaneous tumor; (B-D) immunohistochemical detection of TFPI-2 (arrows) in subcutaneous tumors; (E-F) immunohistochemical detection of BrdU-positive cells (arrowheads) in subcutaneous tumors; (G-H) TUNEL staining for apoptotic cells (arrowheads) in subcutaneous tumors; (I-J) anti-TFPI-2 IgG immunohistochemistry of metastatic lung tumors. Boxed area in I is magnified an additional 2.5-fold in panel J. PC indicates peripheral cells (D,F,H). CC indicates core cells (C,E,G). Original magnifications: × 250 (A-B); × 1000 (I); × 2500 (C-H,J). Hitendra Singh Chand et al. Blood 2004;103:1069-1077 ©2004 by American Society of Hematology

Qualitative PCR analyses of cellular DNA obtained from various tissues by microdissection. Qualitative PCR analyses of cellular DNA obtained from various tissues by microdissection. Cellular DNA used as a template in these PCR reactions was obtained from microdissected cells as described in “Materials and methods,” and PCR products resolved electrophoretically in 1.2% agarose gels. Cellular DNA from mouse gastric and lung cells were processed in an identical manner to exclude the possibility that the human-specific primer pair cross-reacted with mouse DNA. Hitendra Singh Chand et al. Blood 2004;103:1069-1077 ©2004 by American Society of Hematology

Real-time quantitative RT-PCR analysis of murine VEGF gene expression in tumors. Real-time quantitative RT-PCR analysis of murine VEGF gene expression in tumors. (A) Melting curve analysis of the VEGF and GAPDH amplicons. Distinct melting curves of VEGF (dotted line) and GAPDH (solid line) are shown together with controls. (B) Relative efficiency plot of VEGF and GAPDH. The ΔCT (difference in CT values of VEGF and GAPDH) were calculated for each cDNA dilution. (C) Murine VEGF gene expression levels in MT-1080, QT-1080, and WT-1080 tumor samples. Mouse liver RNA (black bar) was used as a positive control. Each column represents the average of 3 amplification reactions (error bars represent standard deviation) performed on a single cDNA sample reverse-transcribed from RNA derived from each tumor sample. Samples MT-2T, MT-3T, and MT-6T are representative MT-1080 tumors (white bars). Samples WT-3T, WT-5T, and WT-11T are representative WT-1080 tumors (dark grey bars), while samples QT-1T, QT-5T, and QT-7T are representative QT-1080 tumors (light gray bars). (D) Agarose gel analyses of PCR products obtained following specific amplification of murine VEGF (top) and murine GAPDH (bottom) amplicons. * indicates negative controls lacking reverse transcriptase in first-strand cDNA synthesis. Hitendra Singh Chand et al. Blood 2004;103:1069-1077 ©2004 by American Society of Hematology