Article Figures & Data
Figures
- FIGURE 1.
CTGF mRNA expression in human blood γδ-T cells. The lane numbers in A represent the corresponding lanes in B and the bar graphs in C. The time points indicated below the bar graph (C) also apply to the corresponding lanes shown in A and B. A, Freshly isolated human blood γδ-T cells (lanes 1–9) and fibroblast cells (lanes 10 and 11) were cultured in the absence (−) or in the presence (+) of TGF-β1 (1 ng/ml)/IL-15 (10 ng/ml). Since Colo 849 fibroblast cell line expresses CTGF constitutively (Fig. 4), one time point was selected (48 h) and used as a positive control. As described in Materials and Methods, the cells were harvested at different time points (0, 4, 8, 24, and 48 h), total RNA was isolated, and RT-PCR was performed on each sample. The PCR products of 521 and 219 bp represent CTGF and β-actin mRNA, respectively. A markedly elevated CTGF mRNA signal was achieved within 48 h after stimulation of human blood γδ-T cells with IL-15/TGF-β1 (lanes 3, 5, 7, and 9). B, β-Actin demonstrated that equivalent quantities of RNA were used for each sample. C, The ordinate demonstrates the ratio of CTGF/β-actin PCR products. Data are representative of four experiments that gave similar results.
- FIGURE 2.
CTGF mRNA expression in Loucy γδ-T cell line. The lane numbers in A represent the corresponding lanes in B and the bar graphs in C. The time points indicated below the bar graph (C) also apply to the corresponding lanes shown in A and B. A, Loucy γδ-T cells (lanes 1–9) and fibroblast cells (lanes 10 and 11) were cultured in the absence (−) or the presence (+) of TGF-β1 (1 ng/ml)/IL-15 (10 ng/ml). Since the Colo 849 fibroblast cell line expresses CTGF constitutively (Fig. 4), one time point was selected (48 h) and used as a positive control. As described in Materials and Methods, the cells were harvested at different time points (0, 4, 8, 24, and 48 h), total RNA was isolated, and RT-PCR was performed on each sample. The PCR products of 521 and 219 bp represent CTGF and β-actin mRNA, respectively. An increased CTGF mRNA expression compared with nonstimulated cells was observed within 48 h after stimulation of Loucy γδ-T cells with IL-15/TGF-β1 (lanes 3, 5, 7, and 9). B, β-Actin demonstrated that equivalent quantities of RNA were used for each sample. C, The ordinate demonstrates the ratio of CTGF/β-actin PCR products. Data are representative of four experiments that gave similar results.
- FIGURE 3.
CTGF mRNA expression in human CD4+ αβ-T cells. The lane numbers in A represent the corresponding lanes in B and the bar graphs in C. The time points indicated below the bar graph (C) also apply to the corresponding lanes shown in A and B. A, Freshly isolated human CD4+ αβ-T cells (lanes 1–9) and Colo 849 fibroblast cell line (lanes 10 and 11) were cultured in the absence (−) or the presence (+) of TGF-β1 (1 ng/ml)/IL-15 (10 ng/ml). As described in Materials and Methods, the cells were harvested at different time points (0, 4, 8, 24, and 48 h), total RNA was isolated, and RT-PCR was performed on each sample. The PCR products of 521 and 219 bp represent CTGF and β-actin mRNA, respectively. CD4+ αβ-T cells did not express CTGF mRNA at any time point tested. Since fibroblast cells express CTGF constitutively (Fig. 4), they were cultured for 48 h and used as a positive control. B, β-Actin demonstrated that equivalent quantities of RNA were used for each sample. C, The ordinate demonstrates the ratio of CTGF/β-actin PCR products. Data are representative of three experiments that produced similar results.
- FIGURE 4.
CTGF mRNA expression in the Colo 849 fibroblast cell line. The lane numbers in A represent the corresponding lanes in B and the bar graphs in C. The time points indicated below the bar graph (C) also apply to the corresponding lanes shown in A and B. A, Fibroblast cells (lanes 1–9) were cultured in the absence (−) or the presence (+) of TGF-β1 (1 ng/ml)/IL-15 (10 ng/ml). As described in Materials and Methods, the cells were harvested at different time points (0, 4, 8, 24, and 48 h), total RNA was isolated, and RT-PCR was performed on each sample. The PCR products of 521 and 219 bp represent CTGF and β-actin mRNA, respectively. CTGF could be expressed constitutively in both nonstimulated and stimulated fibroblast cells (Colo 849). B, β-Actin demonstrated that equivalent quantities of RNA were used for each sample. C, The ordinate demonstrates the ratio of CTGF/β-actin PCR products. Data are representative of three experiments that produced similar results.
- FIGURE 5.
Detection of CTGF protein expression. The lane numbers in A represent the corresponding lanes in B and the bar graphs in C. Lanes 2, 4, 6, and 8 represent nonstimulated cells, whereas lanes 3, 5, 7, and 9 represent stimulated cells. A, Cell lysates used for Western blotting were extracted from 2 × 106 stimulated freshly isolated human CD4+ αβ-T cells (lanes 2 and 3), freshly isolated human blood γδ-T cells (lanes 4 and 5), Loucy γδ-T cells (lanes 6 and 7), and Colo 849 fibroblast cells (lanes 8 and 9). All cell types were cultured in either the absence (−) or the presence (+) of TGF-β1 (1 ng/ml)/IL-15 (10 ng/ml) for 96 h. An increased level of CTGF protein (38 kDa) compared with the nonstimulated cells (lanes 4 and 6, respectively) was detected by freshly isolated human blood γδ-T cells (lane 5) and Loucy γδ-T cells (lane 7) after stimulation with IL-15/TGF-β1. Fibroblast cells synthesize CTGF protein constitutively (lanes 8 and 9). CD4+ αβ-T cells did not synthesize CTGF protein (lanes 2 and 3). The arrows indicate the sizes of the proteins. B, β-Actin demonstrated that equivalent quantities of total protein were used for each sample. C, The ordinate demonstrates the ratio of CTGF/β-actin protein synthesis. Western blots are representative of four experiments that gave similar results. M, Prestained protein marker (lane 1); CD4+, CD4+ αβ-T cells; h-γδ, human γδ-T cells; Loucy, γδ-T cell line; Colo 849, fibroblast cell line.
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