|
|
Blood, Vol. 105, Issue 12, 4649-4656, June 15, 2005
Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation
Blood Morisada et al.
105: 4649
Supplemental materials for: Morisada et al, Vol 105, Issue 12, 4649-4656
Files in this Data Supplement:
- Figure S1. Restricted expression of podoplanin in LECs (JPG, 42 KB)
-
Serial cross-sections of Figure 1H-L were stained with anti-CD31 (red) and antipodoplanin mAbs (green). Note that the expression of podoplanin is restricted in lymphatic vessels (LV) but not in blood vessels (BV). Scale bars indicate 20 µm. Serial cross-sections of Figure 1H-L were reacted with anti–PECAM-1/CD31 and antipodoplanin mAbs at 4°C overnight, followed by staining with secondary antibody Alexa 488–conjugated anti-hamster IgG and Alexa 546–conjugated goat anti-rat IgG, respectively. After being washed with PBS 5 times, samples were analyzed using an FV1000 confocal microscope with UPlan Apo 20×/0.70 (Olympus, Tokyo, Japan).
- Figure S2. Expression of Ang's and VEGFs in LECs, BECs, and OP9 cells (JPG, 50 KB)
-
(A) Expression of Ang-1 and Ang-2 in LECs and BECs (R1 and R2 fractions in Figure 4B, respectively). Note that the Ang-2 is expressed in both LECs and BECs, and that it is more abundant in LECs than in BECs. In contrast, no expression of Ang-1 is detected in either LECs or BECs. (B) FACS analysis of the distribution of LYVE-1+ cells conducted 7 days after starting the coculture with LECs and OP9 stromal cells. (C) Expression of Ang-1, Ang-2, VEGF-C, and VEGF-D in OP9 stromal cells cocultured with (R1 fraction, panel B) or without LECs (OP9). Expression of GAPDH was used as an internal control. mRNA from nonsorted cells without reverse-transcriptase treatment was used as a negative control, indicated as RT(-). For flow cytometry, cells were harvested in dissociation buffer. After treatment with anti–mouse CD16/CD32 Fc receptor (FcR), all cells were stained with biotinylated ALY7 and then reacted with allophycocyanin-conjugated streptavidin to visualize LYVE-1+ cells. An RNeasy Mini Kit (Qiagen, Hilden, Germany) was used to extract RNA from sorted LECs (R1 fraction in Figure 4), BECs (R2 fraction in Figure 4), and OP9 cells (R1 fraction in panel B) after coculturing with LECs and native OP9 stromal cells. RNA was reverse-transcribed with an Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA), and the product was used for further analysis. The sequences of the primers for RT-PCR were as follows: (1) angiopoietin-1, sense: 5´-CAGTGGCTGCAAAAACTTGA -3´; antisense: 5´-TCTGCACAGTCTCGAAATGG-3´; (2) angiopoietin-2, sense: 5´-CACACTGACCTTCCCCAACT-3´; antisense: 5´-TGGTGTCTC-TCAGTGCCTTG-3´; (3) VEGF-C, sense: 5´-GAGCTGATGTCTGTCCTGTACC-3´; antisense: 5´-GGTTTGGGGCCTTGTGAGAGAG-3´; (4) VEGF-D, sense: 5´-GTGTACTTGGTGCAGGGCTTCAGG-3´; antisense: 5´-GGCACTAACTCGG-GCACTGATGTC-3´; PCR products were separated on a 1.2% agarose gel and stained with ethidium bromide.
- Figure S3. Expression of Prox1 in LYVE-1+ cells on OP9 stromal cells (JPG, 43 KB)
-
Immunohistochemical staining of an LEC colony on OP9 feeder cells. (A) LYVE-1 (green). (B) Prox1 (red). (C) TOTO3 (blue). (D) Merged image of panels A and B. Note that LYVE-1+ cells forming the round colony on OP9 feeder cells express Prox1 at the position of nuclei. The cultured cells were fixed in 4% PFA at 4°C for 10 minutes and then washed with PBS 5 times. After cells were treated for 5 minutes with PBS containing 0.1% Triton-X, the cells were reacted with biotinylayed ALY7 and anti-Prox1 antibody (RELIA Tech, Braunschweig, Germany) at 4°C. Expression of LYVE-1 and Prox1 was detected by reacting with Alexa 488–conjugated streptavidin and Alexa 546–conjugated goat antirabbit antibody, respectively. Stained cells were visualized using an FV1000 confocal microscope with UPlan Apo 20×/0.70.
|
|
