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Blood, Vol. 111, Issue 9, 4571-4579, May 1, 2008
Inhibition of endogenous TGF-β signaling enhances lymphangiogenesis
Blood Oka et al.
111: 4571
Supplemental materials for: Oka et al
Files in this Data Supplement:
- Figure S1. Transduction of TGF-β signals in HUVECs (JPG, 96 KB)
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(A) Immunoblotting of phospho-Smad2 after TGF- or TR-I inhibitor treatment for 1 h. HUVECs were treated with TGF-1 (shown as TGF-), TR-I inhibitors (LY364947 or SB431542, shown as Inhib1 or Inhib2, respectively), or TR-I inhibitors with TGF-1, and subjected to immunoblot analysis using phospho-Smad2 antibody (upper panel) and Smad2/3 antibody (lower panel). (B and C) Quantitative real-time PCR of Smad7 and PAI-1. HUVECs were treated as in (A), and expression of Smad7 and PAI-1 mRNAs was determined at 1 h and 24 h after stimulation, respectively. **p<0.01, ***p<0.001. (D) Regulation of growth of HUVECs by TGF- and TR-I inhibitors. HUVECs were seeded at a density of 2 x103 cells/well in 96-well plates, and cells were treated or not with TGF-1 (1 ng/ml) or TR-I inhibitor (3 µM). Growth of cells was determined by WST assay in triplicate at day 2. Error bars represent standard deviations. (E) Autocrine TGF- signaling in HUVECs. Expression of TGF-1 in HUVECs was determined by real-time PCR. Error bars represent standard deviations. p***<0.001.
- Figure S2. Autocrine TGF-β signaling in HDLECs (JPG, 63.2 KB)
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Expression levels of TGF-2 and TGF-3 mRNAs in HDLECs were determined by real-time PCR as in Fig. 1E. n.s., not significant.
- Figure S3. Regulation of the expression of Prox1 and LYVE-1 by TGF-β1 or TβR-I inhibitors in HDLECs and HUVECs (JPG, 98.4 KB)
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Expression levels of Prox1 and LYVE-1 mRNAs were analyzed in HDLECs (A, B) or HUVECs (C, D) by real-time PCR at 24 h after treatment with TGF-1 or TR-I inhibitors (LY364947 or SB431542, shown as Inhib1 or Inhib2, respectively). Values were normalized to amounts of GAPDH mRNA. Error bars represent standard deviations. *p<0.05, **p<0.01, and ***p<0.001.
- Figure S4. Inhibition of lymphangiogenesis by TGF-β1 in the MIA PaCa-2 xenograft model (JPG, 45.5 KB)
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Effects of TGF-1 on lymphangiogenesis were examined in a xenograft model using GFP- or active TGF-1-expressing MIA PaCa-2 cells. MIA PaCa-2 cells were subcutaneously inoculated in BALB/c nude mice as described in Figure 7. Bars 50 µm. (A) Expression of TGF-1 in MIA PaCa-2 cells determined by quantitative RT-PCR. *p<0.05 (B) Immunostaining of the GFP- (Ctrl) or active TGF-1-expressing (TGF-) MIA PaCa-2 xenograft sections by LYVE-1 (shown in green). (C) LYVE-1-positive areas in the GFP- or active TGF-1-expressing MIA PaCa-2 xenograft sections were determined (n=3 for each group). Error bars represent standard errors. ***p<0.01.
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