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Additional Supporting Information may be found in the online version of this article.

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IJC_24040_sm_suppfigure1.tif2130KSupporting Figure 1: (A) Effects of recombinant CXCL12 on CXCL8 production from AsPC-1 PaCa cell line. To determine the effects of CXCL12 on CXCL8 production from a distinct PaCa cell line, AsPC-1 cells were seeded at a density of 2 × 105 cells/mL into a 24-well plate containing medium with 10% FCS and cultured overnight. Medium was then exchanged, and cells were cultured for a further 48 h treated with or without CXCL12 (20 ng/mL). The concentration of CXCL8 was measured using ELISA kit as described in Materials and Methods. Values are expressed as mean ± SD. **, P < 0.05. (B) Effects of recombinant CXCL8 on CXCL12 production from fibroblast cells. To determine the effects of CXCL8 on CXCL12 production from fibroblast cells, fibroblast cells were cultured treated with or without CXCL8 (20 ng/mL) for 48 h and the concentration of CXCL12 in the supernatants was measured by ELISA kit in the same way. Values are expressed as mean ± SD. n. s., no significant difference. (C) To determine the effects of co-culture with a distinct PaCa cell line on CXCL12 production from fibroblast cells, we co-cultured fibroblasts with AsPC-1 cells using a double chamber method (AsPC-1; 5×104 cells into inserts with 0.4-μm pores [BD Biosciences], fibroblast; 2×105 cells into 24-well plates). After 48 h incubation, the concentration of CXCL12 in supernatant was measured by ELISA kit in the same way. Values are expressed as mean ± SD. **, P < 0.05
IJC_24040_sm_suppfigure2.tif1501KSupporting Figure 2: Effects on MMP-2 and MMP-9 activities in HUVEC or PaCa cells following treatment with CXCL12. (A) Zymography analysis of HUVEC. HUVEC cells were seeded at a density of 2 × 106 cells/3mL into 60 mm dish and cultured overnight. Medium were then exchanged and cells were cultured for a further 24 h treated with or without recombinant CXCL12 (100 ng/ml). Using these supernatants and cell lysates, Zymography assay was performed. The samples were mixed with sample buffer without reducing agent or heating, and loaded into Zymogram gels (Invitrogen, Carlsbad, CA). After the electrophoresis with constant voltage, the gel was treated with Zymogram Renaturing Buffer (Invitrogen) and Zymogram Developing Buffer (Invitrogen) according to their instructions. Finally the gel was stained with SimplyBlue Safestain (Invitrogen) according to the instruction. (B) Zymography analysis of PaCa cells. In the same way, the supernatants of AsPC-1, Panc-1, and BxPC-3 cells treated with or without CXCL12 were collected and Zymography analysis was performed as described above.

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