Figure 2.
Figure 2. (A, i-ii) TPO induced expression of GPVI on UT-7/EPO Mpl cells. Total RNA was extracted from cells treated with 10 ng/mL TPO for 3, 5, or 9 days or with 10 μM 5-aza-dC for 48 hours. GPVI (i) and GAPDH (ii) mRNA were then amplified by RT-PCR. By this method, GPVI mRNA was detected in cells treated with TPO for 5 or 9 days or in cells treated with 5-aza-dC. (iii-v) Surface expression of GPVI by UT-7/EPO Mpl cells treated with TPO, as determined by flow cytometry. — represents binding of the murine monoclonal antihuman GPVI antibody 204-11; --- corresponds to staining with fluorescein isothiocyanate-labeled secondary antibody alone. (iii) No binding was observed when UT-7/EPO Mpl are cultured in the presence of EPO. (iv) Weak, but reproducible, binding was observed following culture of UT-7/EPO Mpl in the presence of TPO for 9 days. (v) Intense binding is observed when UT-7/TPO are cultured in the presence of TPO. These results are representatives of identical findings made in 3 independent experiments. (B) Methylation-specific PCR of the GP6 promoter CpG island. Reverse primers were designed to distinguish the unmethylated (left) or methylated (right) DNA. The methylation-negative primer dmsp-R1 includes the eighth and ninth CpG sites converted to TG; in the methylation-positive primer msp-R2, the ninth CpG site is conserved as CG. The GP6 product (170 bp) is obtained using dmsp-R1 only when the cells are treated with TPO. (C) Methylation status of GP6 CpG sites in the product amplified with dmsp-R1 and shown in panel B. DNA was isolated from UT-7/EPO Mpl cells stimulated with TPO for 3 days or 5-aza-dC for 48 hours and treated with sodium bisulfite. PCR was performed with the same forward primer used in Figure 1B and dmsp-R1 used as the reverse primer. Amplified DNA was cloned into pGEM-T easy vector, and the methylation status was determined by DNA sequencing. • indicates methylated CpG sites; ○, unmethylated sites; and, sites contained within the reverse primers. Numbers at the top correspond to the same CpG sites shown in Figure 1A.

(A, i-ii) TPO induced expression of GPVI on UT-7/EPO Mpl cells. Total RNA was extracted from cells treated with 10 ng/mL TPO for 3, 5, or 9 days or with 10 μM 5-aza-dC for 48 hours. GPVI (i) and GAPDH (ii) mRNA were then amplified by RT-PCR. By this method, GPVI mRNA was detected in cells treated with TPO for 5 or 9 days or in cells treated with 5-aza-dC. (iii-v) Surface expression of GPVI by UT-7/EPO Mpl cells treated with TPO, as determined by flow cytometry. — represents binding of the murine monoclonal antihuman GPVI antibody 204-11; --- corresponds to staining with fluorescein isothiocyanate-labeled secondary antibody alone. (iii) No binding was observed when UT-7/EPO Mpl are cultured in the presence of EPO. (iv) Weak, but reproducible, binding was observed following culture of UT-7/EPO Mpl in the presence of TPO for 9 days. (v) Intense binding is observed when UT-7/TPO are cultured in the presence of TPO. These results are representatives of identical findings made in 3 independent experiments. (B) Methylation-specific PCR of the GP6 promoter CpG island. Reverse primers were designed to distinguish the unmethylated (left) or methylated (right) DNA. The methylation-negative primer dmsp-R1 includes the eighth and ninth CpG sites converted to TG; in the methylation-positive primer msp-R2, the ninth CpG site is conserved as CG. The GP6 product (170 bp) is obtained using dmsp-R1 only when the cells are treated with TPO. (C) Methylation status of GP6 CpG sites in the product amplified with dmsp-R1 and shown in panel B. DNA was isolated from UT-7/EPO Mpl cells stimulated with TPO for 3 days or 5-aza-dC for 48 hours and treated with sodium bisulfite. PCR was performed with the same forward primer used in Figure 1B and dmsp-R1 used as the reverse primer. Amplified DNA was cloned into pGEM-T easy vector, and the methylation status was determined by DNA sequencing. • indicates methylated CpG sites; ○, unmethylated sites; and, sites contained within the reverse primers. Numbers at the top correspond to the same CpG sites shown in Figure 1A.

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