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Fig. 5. PPI-2458 induced growth inhibition on human fibroblast-like synoviocytes from rheumatoid arthritis patients (HFLS-RA) is not mediated by cytotoxicity and is completely reversible. (A) HFLS-RA (1 × 10⁴ cells) were incubated either in drug-free medium (■) or medium containing 10- ⁷ M PPI-2458 (dotted bars) for 7 days, stained with fluorescence-conjugated annexin V, and analyzed by flow cytometry. (B) HFLS-RA (8 × 10³ cells) were incubated with increasing concentrations (10- ⁹, 10-⁸, and 10-⁷ M) of PPI-2458 for 7 days. After 7 days, HFLS-RA were washed once with drug-free medium and incubated in either drug-free medium (filled bars) or PPI-2458 containing medium (dotted bars) for another 7 days. For the final 24 h, 1 mCi of [³H]thymidine per well was added, and proliferation was determined by [³H]thymidine incorporation.

Fig. 6. Western blot of methionine aminopeptidase (MetAP)-2, MetAP-1, and proliferating cell nuclear antigen (PCNA) expression in PPI-2458 sensitive HFLS-RA (A) and human umbilical vein endothelial cells (HUVEC) (C), and PPI-2458-resistant normal human dermal fibroblasts from adults (NHDF-Ad) (B). HFLS-RA (8 × 10³) and NHDF-Ad were incubated for 7 days, 8 × 10³ HUVEC for 4 days, with increasing concentrations (10- ¹²-10- ⁷ M) of PPI-2458, as described in Materials and Methods. After cell lysis, 20 m g of cellular protein was immunoblotted with specific primary antibody to each protein and visualized with streptavidin-conjugated horseradish peroxidase.

Fig. 7. Dexamethasone, but not PPI-2458, inhibits the secretion of IL-6 (A) and vascular endothelial growth factor (VEGF) (B) from tumor necrosis factor a(TNF-a )- (1 ng/ml) and lipopolysaccharide (LPS)-stimulated (10 m g/ml) HFLS-RA in vitro. Quantitation of IL-6 and VEGF in tissue culture supernatants was performed as described in Materials and Methods. Results represent the mean ± SEM (n = 3). *P <0.05 and **P [lt] 0.01 compared to TNF-a-stimulated (A) or LPS-stimulated HFLS-RA (B). ** P [lt] 0.01 compared to unstimulated HFLS-RA (B).