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SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure 1. Regulation of IL-8 and its receptors, CXCR1 and CXCR2, by TNF-α
and 9-cisRA. (a-c) Quantitation of PCR-bands shown in Figure 1b for CXCR1 (a), CXCR2 (b),
and IL-8 (c) using NIH Image J software. (d,e) Upregulation of IL-8 expression by TNF-α (5
ng/ml) (e) and 9-cisRA (1 µM) (e) was also confirmed using quantitative real-time RT-PCR
(qRT-PCR).
Supplemental Figure 2. IL-8 downregulates p57Kip2 expression by downregulating PROX1. (a)
PROX1 expression was progressively reduced in primary LECs after 12 hours of IL-8 treatment
and recovered 24-hour post treatment. In comparison, p57Kip2 is downregulated at 18 hours. (b)
Adenoviral expression of PROX1 in LECs prior to IL-8-treatment prevented the IL-8-induced
downregulation of p57Kip2 in LECs. These data were obtained from a repeated experiment of
Figure 3a and c.
Supplemental Figure 3. IL-8 activates lymphatic vessel ingrowth in matrigel plug assays.
Vascular analysis was performed to quantify lymphatic vessels found in the matrigels shown in
Figure 4a.
Supplemental Figure 4. Tumor growth curves of colon cancer cells in mice. (A) Human
(HCT116) and (B) mouse (CT26) colon cancer cells were subcutaneously implanted into nude
vs. K14-hIL8/nude mice (n = 5, each group) and their tumor size (mm3) was measured over 2~3
weeks. (C) Similarly, mouse colon cancer (CT26) cells were grafted into the skin of CXCT2 wildtype (WT) or knockout (KO) mice (n = 5, each group) and tumor growth was monitored for two
weeks. *, p < 0.05).
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Supplemental Figure 5. High resolution Hematoxylin and Eosin (H&E) staining show the
absence of considerable immune cell infiltration into the surgically wounded area for induction of
secondary lymphedema. Bar, 100 µm.
Supplemental Figure 6. Comparable macrophage infiltration into the lymphedema area in the
tails of wild-type (WT) vs. K14-hIL8 transgenic mice. Frozen tail sections at day 29 post-surgery
were stained for mouse macrophage-specific antigen F4/80 and representative images are
shown. Note the comparable numbers of F4/80-positive cells in both groups of mice. Bar, 50
µm.
Supplemental Figure 7. Scarce neutrophil infiltration into the lymphedema area in the tails of
wild-type (WT) and K14-hIL8 transgenic mice. Frozen tail sections of each group at day 29 postsurgery were stained for mouse neutrophil-specific antigen LY-6G and representative images
are shown. Only a small number of neutrophils were detected in both groups of mice (white
arrow). Bar, 50 µm.
Supplemental Figure 8. Enhanced lymphangiogenesis in secondary lymphedema in the tail of
K14-hIL8 transgenic (TG) compared to wild-type mice (WT). Frozen tail sections from both mice
at 29 days post-surgery were immuno-stained for a lymphatic-specific marker, podoplanin.
White arrows mark podoplanin-positive lymphatic vessels in the wounded regions of the tails.
Bar, 50 µm.
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