American Society of Hematology

Fig. 3.

$Fig. 3. Endoglin induction by KLF6 after transient transfection. / (A) Analysis of endogenous endoglin expression in HeLa cells by flow cytometry. HeLa cells were cotransfected with 4 μg pCIneo KLF6 (KLF6), pCIneo, pcDNA3-EGR1 (EGR1), orpcDNA3, and 1 μg pEGFP-C2 (GFP), as indicated. Transfected and untransfected cells were stained with the mouse monoclonal antibody P4A4 (antiendoglin), followed by incubation with FluoroLink Cy5-labeled goat-anti–mouse IgG. Cells were washed with PBS, and their fluorescence was estimated with a FACSVantage by detecting the Cy5 (absorbance at 649 nm, emission at 670 nm) and the green fluorescence protein (absorbance at 488 nm, emission at 507 nm) fluorochromes. Transfected cells were previously sorted using the green fluorescence protein as a transfection marker. Surface expression of endoglin was measured by detecting the fluorescence of Cy5. Numbers in the upper right corner indicate the mean fluorescence intensity from endoglin. In parentheses are shown the fold induction values for KLF6 (1.5) and EGR1 (1.1) with respect to the corresponding empty vectors. Staining with an irrelevant antibody (control antibody) was also included as a negative control. The broken vertical line indicates the fluorescence intensity of the negative control. Shown is 1 of 5 representative experiments that gave similar results. (B-C) RT-PCR analysis of endoglin and GAPDH mRNA levels in mock versus KLF6-transfected HeLa (B) and M1 (C) cells. Cells were transfected with 4 μg empty vector (M) or pCIneo-KLF6 (K). Aliquots from the PCR reaction were isolated after the indicated number of cycles and were analyzed by electrophoresis in 5% Nu-Sieve agarose gels. Bar graphs representing densitometry quantification of endoglin/GAPDH ratios from cells transfected with empty vector (■) orpCIneo-KLF6 (▪) are shown on the right. (D) Induction of endogenous endoglin expression after transfection with KLF6. BAECs were grown on 10-cm plastic plates and transiently transfected withpCIneo empty vector (control) or pCIneo-KLF6plasmid (5 or 10 μg). Cells were harvested 24 hours later, total RNA was extracted, and synthesis of cDNA was performed. Comparative quantitation of endoglin mRNA to GAPDH was analyzed with real-time RT-PCR. Fluorescence signals were analyzed during each of 40 cycles (denaturation 15 seconds at 95°C, annealing 15 seconds at 56°C, and extension 40 seconds at 72°C). Relative expression was calculated using the comparative threshold cycle (CT) method. CT indicates the fractional cycle number at which the amplified gene amounts to a fixed threshold within the linear phase of amplification. Median CT of triplicate measurements was used to calculate ΔCT as the difference in CTfor endoglin and GAPDH. ΔCT for each sample was compared to the control CT and expressed as ΔΔCT. Data are expressed as fold induction of endoglin (normalized for GAPDH), compared with vector-transfected cells, with the formula 2−Δ (CT). Shown is 1 of 2 representative experiments.$

Endoglin induction by KLF6 after transient transfection.

(A) Analysis of endogenous endoglin expression in HeLa cells by flow cytometry. HeLa cells were cotransfected with 4 μg pCIneo KLF6 (KLF6), pCIneo, pcDNA3-EGR1 (EGR1), orpcDNA3, and 1 μg pEGFP-C2 (GFP), as indicated. Transfected and untransfected cells were stained with the mouse monoclonal antibody P4A4 (antiendoglin), followed by incubation with FluoroLink Cy5-labeled goat-anti–mouse IgG. Cells were washed with PBS, and their fluorescence was estimated with a FACSVantage by detecting the Cy5 (absorbance at 649 nm, emission at 670 nm) and the green fluorescence protein (absorbance at 488 nm, emission at 507 nm) fluorochromes. Transfected cells were previously sorted using the green fluorescence protein as a transfection marker. Surface expression of endoglin was measured by detecting the fluorescence of Cy5. Numbers in the upper right corner indicate the mean fluorescence intensity from endoglin. In parentheses are shown the fold induction values for KLF6 (1.5) and EGR1 (1.1) with respect to the corresponding empty vectors. Staining with an irrelevant antibody (control antibody) was also included as a negative control. The broken vertical line indicates the fluorescence intensity of the negative control. Shown is 1 of 5 representative experiments that gave similar results. (B-C) RT-PCR analysis of endoglin and GAPDH mRNA levels in mock versus KLF6-transfected HeLa (B) and M1 (C) cells. Cells were transfected with 4 μg empty vector (M) or pCIneo-KLF6 (K). Aliquots from the PCR reaction were isolated after the indicated number of cycles and were analyzed by electrophoresis in 5% Nu-Sieve agarose gels. Bar graphs representing densitometry quantification of endoglin/GAPDH ratios from cells transfected with empty vector (■) orpCIneo-KLF6 (▪) are shown on the right. (D) Induction of endogenous endoglin expression after transfection with KLF6. BAECs were grown on 10-cm plastic plates and transiently transfected withpCIneo empty vector (control) or pCIneo-KLF6plasmid (5 or 10 μg). Cells were harvested 24 hours later, total RNA was extracted, and synthesis of cDNA was performed. Comparative quantitation of endoglin mRNA to GAPDH was analyzed with real-time RT-PCR. Fluorescence signals were analyzed during each of 40 cycles (denaturation 15 seconds at 95°C, annealing 15 seconds at 56°C, and extension 40 seconds at 72°C). Relative expression was calculated using the comparative threshold cycle (C_T) method. C_T indicates the fractional cycle number at which the amplified gene amounts to a fixed threshold within the linear phase of amplification. Median C_T of triplicate measurements was used to calculate ΔC_T as the difference in C_Tfor endoglin and GAPDH. ΔC_T for each sample was compared to the control C_T and expressed as ΔΔC_T. Data are expressed as fold induction of endoglin (normalized for GAPDH), compared with vector-transfected cells, with the formula 2^{−Δ (CT)}. Shown is 1 of 2 representative experiments.

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