Xiangde Liu, Amy Nelson, Xingqi Wang, MahaFarid, Yoko Gunji, Jun Ikari, ShunIwasawa, HeshamBasma, Carol Feghali-Bostwick, Stephen I.

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1 Online Data Supplement Vitamin D Modulates PGE 2 Synthesis and Degradation in Human Lung Fibroblasts Xiangde Liu, Amy Nelson, Xingqi Wang, MahaFarid, Yoko Gunji, Jun Ikari, ShunIwasawa, HeshamBasma, Carol Feghali-Bostwick, Stephen I. Rennard

2 Supplement Materials and Methods Collagen gel contraction assay Gels were prepared using a previously published method (18). Briefly, distilled water, 4 X concentrated DMEM and RTTC were mixed so that the final mixture resulted in a physiological ionic strength, that is, 1 X DMEM and a ph of 7.4. Cell pellets were suspended with serum free DMEM (SF-DMEM) at a density of 10 7 cells/ml and then mixed with the RTTC solution mentioned above so that the final cell density in the collagen solution was 3 x 10 5 cells/ml, and the final concentration of collagen was 0.75 mg/ml. Aliquots of the mixture were then cast into each well of 24-well tissue culture plate (0.5ml/well). After 20 minutes at room temperature during which gelation occurs, the gels were released into 60mm tissue culture dishes (three gels each dish), which contained 5ml of freshly prepared SF-DMEM with or without vitamin Ds as designed. The gels were then incubated at 37 C in a 5% CO 2 atmosphere for 5 days as indicated, and the area of each gel was measured with an Optimax V Image Analyzer (Optimax, Burlington, MA) daily. Data were expressed as the percentage of the size compared to original gel size. Supplement Figure 1. Effect of vitamin Ds on PGE 2, fibronectin, TGF-ß1 and VEGF release by HFL-1 cells cultured in three-dimensional collagen gels. HFL-1 cells were cast into the collagen gelsand released into SF-DMEM containing EOH (1:1000), 1µM vitamin D, 25(OH)D or 1,25(OH) 2 D.After 2 days culture, medium was harvested for quantification of PGE 2, fibronectin, TGF-ß1 or VEGF as described in the Materials and Methods. Vertical axes: protein amount adjusted by cell number; horizontal axes: treatment. Data presented are one representative from 4 separate experiments. ** p<0.05. Supplement Figure 2. Effect of Vitamin D, 25(OH)D and 1,25(OH) 2 D fibroblast-mediated collagen gel contraction. Panel A: Time-dependent

3 effect. HFL-1 cells were cast into collagen gels as described in the materials and methods. After 20 minutes polymerization at room temperature, gels were released into SF-DMEM plus EOH (1:1000), vitamin D (1µM), 25(OH)D (1µM) or 1,25(OH) 2 D (1µM) and allowed to contract for 5 days. Gel size was measured with an image analyzer daily. Vertical axis: gel size expressed as percent of initial size (%); horizontal axis: time (day). Data presented is one representative of 6 separate experiments. Panel B: Concentration-dependent effect. Collagen gels with fibroblasts were released into SF-DMEM containing varying concentrations of vitamin D, 25(OH)D or 1,25(OH) 2 D. Gel size was measured on day 2. Vertical axis: gel size expressed as percent of initial size (%); horizontal axis: concentrations of vitamin Ds (nm). Data presented is one representative of 3 separate experiments. Panel C: Effect of exogenous PGE 2. Collagen gels with fibroblasts were released into SF-DMEM containing varying concentrations of PGE2 witheoh (1:1000), vitamin D (1µM), 25(OH)D (1µM) or 1,25(OH) 2 D (1µM). Gel size was measured on day 2. Vertical axis: gel size expressed as percent of initial size (%); horizontal axis: concentration of PGE 2 (M). Data presented is one representative of 3 separate experiments.* p<0.05, ** p<0.01 compared to no PGE 2 by Two-way ANOVA followed by Bonferroni correction. Panel D: Effect of indomethacin. HFL-1 cells were pre-treated with 1µM indomethacin for 4 hours. Cells were then cast into collagen gels and released into SF-DMEM supplemented with 0, 100nM, 500nM or 1000nM 25(OH)D. Gel size was measured on day 2. Vertical axis: gel size expressed as percent of initial size (%); horizontal axis: concentrations of 25(OH)D (nm). Data presented is one representative of 3 separate experiments. * p<0.05, ** p<0.01 by Two-way ANOVA followed by Bonferroni correction. Supplement Figure 3. Effect of vitamin D on -smooth muscle actin ( -SMA) expression.hfl-1 cells were treated with ethanol (1:1000), 1µM vitamin D, 25(OH)D, 1,25(OH)2D, or 100pM TGF-ß1 for 24 hours. Total cell lysates were harvested and immunoblotted to -SMA and ß-actin as described in the methods.

4 Supplement Figure 4. Effect of VDR-siRNA on the expression of 15-PGDH and mpges-1.hfl-1 cells were transfected with control-sirna or VDR-siRNA. Total cell lysates were harvested and immunoblottedfor VDR, 15-PGDH, mpges-1 and ß-actin as describedin the materials and methods.

5 A. B. C. D. Supplemental Figure 1

6 A. Time-dependent effect C. Effect of exogenous PGE 2 B. Concentration-dependent effect D. Effect of indomethacin Supplement Figure 2

7 Effect on α-sma EOH Vitamin D 25(OH)D 1,25(OH) 2 D TGF-ß1 α-sma ß-actin Supplement Figure 3

8 Con-siRNA VDR-siRNA ß-actin VDR 15-PGDH mpges-1 Supplement Figure 4