<i>Hrg</i> deletion in mice suppresses Lewis Lung tumor vascularity.

<p>(<b>A</b>) Lewis Lung tumors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g003" target="_blank">Figure 3</a> were dissected, sectioned and examined by immunofluorescence microscopy using anti-VEGF receptor antibody (green) to detect blood vessels. DAPI stained nuclei are blue. Magnification bars represent 100 µm. IgG control is shown in bottom panel as negative control. (<b>B</b>) Vessel densities measured as vessels per mm2. Median vessel density: wt 12.33, cd36 null 6.99.</p

Thrombospondin-1 expression in LL2 and B16F1 melanoma cells and tumors.

<p>(<b>A</b>) Lewis Lung (LL2) or B16F melanoma cells were cultured in serum free media for 24 hours (1d) at which point proteins in post culture media (CM) were precipitated by TCA, separated under reducing conditions by SDS/PAGE and analyzed by immunoblot using anti-TSP-1 antibody. TSP-1 monomers were detected at 170 kDa in the media conditioned by LL2 cells, but not B16F1 cells. Purified human HRG and TSP were used as controls. (<b>B</b>) B16F1 melanoma tumor tissue was analyzed by western blot analysis for TSP expression. Intact TSP was not observed at 150 kDa, however possible degredation products were observed around 55 kDa. (<b>C</b>) B16F1 and LL2 tumor tissue was analyzed by RT-PCR for expression of TSP. TSP was detected in both tumor types, approximately 7 fold higher in LL2. (<b>D</b>) Conditioned media was collected from 4 different antibiotic resistant clones of TSR transfected B16F melanoma cells and analyzed by immunoblot as in panel A. Clone 11 expressed abundant anti-TSP reactive material at the appropriate molecular weight of recombinant TSR and was utilized for subsequent tumor studies. (<b>E</b>) TSP knockdown efficiency was analyzed by RT-PCR with statistical significance as indicated; **P<0.05; *P = 0.06. In both instances of TSP knockdown 1 (K1 and K2), reductions in TSP message levels were detected as compared with nontargeted control (NT) cells.</p

TSR transfected B16F1 melanoma cells show enhanced tumor vascularity in <i>cd36</i> null mice and suppressed tumor vascularity in <i>hrg</i> null mice.

<p>(<b>A</b>) Tumors from TSR transfected B16F1 melanoma cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g006" target="_blank">Figure 6</a> were dissected, sectioned and examined by immunofluorescence microscopy using anti-VE-Cadherin antibody (green) to detect blood vessels. DAPI stained nuclei are blue. Magnification bars represent 100 µm. IgG control is shown in bottom panels as negative control. (<b>B</b>). Median vessel density: wt 8.99, cd36 null 16.32. Median vessel density: wt 9.33, hrgp null 5.83. (<b>C</b>) Tumors from lentiviral (NT, K1 and K2) transfected LL2 cells from wildtype and cd36 null mice were examined using anti-VE-Cadherin antibody to detect blood vessels. Vessel densities measured as vessels per mm².</p

<i>Hrg</i> deletion in mice suppresses syngeneic tumor growth.

<p>Lewis Lung carcinoma cells (<b>A</b>) or B16F1 melanoma cells (<b>B</b>) were injected in the backs of <i>hrg</i> null or wild type C57BL/6 mice (50,000 cells/mouse). Tumor volumes were assessed over 16 days following implantation. *P<0.05.</p

<i>Cd36</i> deletion in mice enhances syngeneic tumor growth.

<p>Lewis Lung carcinoma cells (<b>A</b>) or B16F1 melanoma cells (<b>B</b>) were injected in the backs of <i>cd36</i> null or wild type C57BL/6 mice (50,000 cells/mouse). Tumor volumes were assessed over 17 days following implantation. *P<0.05.</p

TSR transfected B16F1 melanoma cells show enhanced tumor growth in <i>cd36</i> null mice and suppressed tumor growth in <i>hrgp</i> null mice.

<p>50,000 cells from a stably transfected B16f1 melanoma cell line (Clone 11) were injected in the backs of <i>cd36</i> null (A) or <i>hrgp</i> null (B) mice. C57BL/6 mice were used as controls. Tumor volumes were assessed at timed points as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g003" target="_blank">3</a>. *P<0.05; **P = 0.08; ***P = 0.06. LL2 cells stabily transfected with nontargeted (NT) or TSP targeted constructs, K1 and K2, constructs were similarly injected subcutaneously onto the backs of wildtype (WT) and cd36 null mice (Null). (<b>C</b>) WT-NT tumors grew smaller than WT-K1 and WT-K2 tumors, with statistically significant differences seen at days 7 and 15 for both NT vs K1 and NT vs K2. (<b>D</b>) No differences were observed between NT, K1 and K1 in cd36 null mice.</p