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cbin10088-sm-0001-SupFig-S1.doc1600K

Figure S1. Expression of VEGF receptors in FRCs. RNA was isolated from FRCs in monoculture or in coculture with ECs in presence or absence of Dex prior to coculture. RNA was then used in RT-PCR for VEGF receptors (Vegf-R1 and NP-1) and the GAPDH control. Expression was normalised to Gapdh expression and expressed as relative expression (band intensity/gapdh band intensity), n = 3. (A) Expression of Vegf-R1 in FRCs. (B) Variable Np-1 expression in FRCs. No significant differences were noted for either receptor.

Figure S2. Expression of Vegf in ECs over 14 days in coculture with FRCs. RNA was isolated from ECs in coculture with FRCs previously exposed to Dex (Coculture EC+Dex) or not (Coculture EC-Dex) or in monoculture (control). RNA was then used in RT-PCR for Vegf isoforms, and Gapdh as a control. Expression was normalised to Gapdh expression and expressed as relative expression (band intensity/gapdh band intensity) ± SD, n = 3. (A) Expression of Vegf120 in ECs. (B) Expression of Vegf164. Similar patterns were seen in all groups but no significant differences between them. A significant reduction in Vegf164 was noted at 48 h for all groups compared to time 0, **P < 0.01. (C) Vegf188 and (D) Vegf144 expression patterns again showed similar patterns with no significant differences; n = 3.

Figure S3. Expression of VEGF receptors in ECs. RNA was isolated from ECs in monoculture or in coculture with FRCs in presence or absence of Dex prior to coculture. RNA was then used in RT-PCR for VEGF receptors (Vegf-R1, Vegf-R2, Np-1 and Np-2) and the Gapdh control. Expression was normalised to Gapdh expression and expressed as relative expression (band intensity/gapdh band intensity) ± SD, n = 3. (A) Vegf-R1 expression, (B) Vegf-R2 expression, (C) Np-1 expression and (D) Np-2 expression. Similar patterns were seen in all groups with little significant difference except for a significant decrease in Np-1 expression in both coculture groups compared to the monoculture control at Day 14. +P < 0.05; n = 3.

Figure S4. Endothelin-1 receptor, ETRA expression in FRCs and ECs. RNA was isolated from ECs and FRCs in monoculture or in coculture. Prior to the start of cocultures, FRCs were in the presence or absence of Dex for 4 days. RNA was then used in RT-PCR for Endothelin-1 (ET-1), its receptors (ETRA and ETRB), and the GAPDH control. Expression was normalised to Gapdh expression and expressed as relative expression (band intensity/Gapdh band intensity) ± SD, n = 3. No significant differences/patterns were seen in Et-1 or Etrb expression in FRCs or ECs (A and C, and, D and F, respectively). Etra expression in FRCs (B) demonstrated a similar drop in all groups at the beginning of culture followed by a steady increase from 8 h to Day 14 but no significant differences were seen between the groups. (E) Etra in ECs demonstrated a similar drop in the first 8 h of culture and sharply increased after 96 h in culture.

cbin10088-sm-0002-SupTable-S1.doc64KTable S1. Primers and experimental conditions.

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