Figure 3

Effect of long-pentraxin 3 (PTX3) overexpression on B16-LS9 cells. (A) RT-PCR analysis of Fgf2 and Fgfr expression in B16-LS9 cells. (B) Western blot analysis of the phosphorylation of FGFR1 and FGFR3 and of the downstream signaling proteins ERK_1/2 and AKT in B16-LS9 cells following 30 min treatment with 30 ng/mL FGF2. (C) RT-PCR analysis of human PTX3 (hPTX3) expression in WT_LS9, mock_LS9, and hPTX3_LS9 cells. Inset: Western blot analysis of PTX3 protein levels in the extracts of the same cells. (D) Western blot analysis of the phosphorylation of FGFR1, FGFR3, FRS2, and ERK_1/2 proteins in WT_LS9, mock_LS9, and hPTX3_LS9 cell extracts. (E) WT_LS9, mock_LS9, and hPTX3_LS9 cells were seeded in 48-well plates at 10⁴ cells/well in medium containing 0.4% FBS. After 24 h (T₀), medium was changed, and cell were counted 24 and 48 h thereafter. Data are the mean ± SEM of three independent experiments in triplicate. (F) Cells were seeded as in (E). At T₀, cells were treated with 30 ng/mL FGF2 and counted 24 h thereafter. Data are the mean ± SEM of three independent experiments in triplicate (G) WT_L69, mock_LS9, and hPTX3_LS9 cells were seeded at 50 cells/cm². After 10 days, cell colonies were stained with crystal violet and quantified by computerized image analysis. Representative images of mock_LS9 and hPTX3_LS9 cell colonies are shown on the right. Data are the mean ± SEM of 15 fields for each triplicate sample. (H) A mechanical wound was performed in WT_LS9, mock_LS9, and hPTX3_LS9 cell monolayers. After 18 h, cell migration at the leading edge of the wound was quantified by computerized image analysis. Representative images of wounded mock_LS9 and hPTX3_LS9 cell monolayers are shown on the right. Data are the mean ± SEM of six microscopic fields. (I) Mock_LS9 and hPTX3_LS9 cells were injected subcutaneously (s.c.) in syngeneic mice at 50,000 cells/graft and tumor growth was measured with calipers. Data are the mean ± SEM (n = 16). (J) Red fluorescent WT_LS9, mock_LS9, and hPTX3_LS9 cells were injected into the bloodstream of 48 hours post fertilization (hpf) zebrafish embryos (80–100 cells/embryo). During the next 3 days, the growth of fluorescent metastases in the tail vascular plexus was quantified by fluorescence microscopy followed by computerized image analysis. Data are the mean ± SEM of three independent experiments (n = 20) and were normalized to metastasis areas at day 1. (K) WT_LS9 cells were injected s.c. in wild-type and transgenic TgN (Tie2-hPTX3) mice (50,000 cells/graft) and tumor growth was measured with calipers. Data are the mean ± SEM (n = 18). (L) WT_LS9 cells were injected into the spleen of wild-type and transgenic TgN (Tie2-hPTX3) mice (20,000 cells/graft). After 14 days, livers were harvested, and metastases were counted. Representative images of harvested livers are shown on the right. Data are the mean ± SEM (n = 5). In (B) and (D), the right panel shows the densitometric analysis of immunoreactive bands normalized to α-tubulin protein levels. *p < 0.05; **p < 0.01, Student’s t-test (F,L), one-way (E,H,J) and two-way (I,K) analysis of variance.