Table 1.
Expression of endoglin in endothelial cells, tumor cells and tumors, expressed as threshold cycle values (Ct) obtained by qRT-PCR in comparison to reference gene.
Figure 1.
Transfection of endothelial cells with siRNA against endoglin resulted in reduced mRNA and protein levels.
(A) qRT-PCR analysis of endoglin mRNA level after the transfection of endothelial cells HMEC-1 with 3 different siRNA molecules (h_siRNA 529, h_siRNA 240, h_siRNA 241). (B) qRT-PCR analysis of endoglin mRNA level after the transfection of endothelial cells 2H11 with 3 different siRNA molecules (m_siRNA 868, m_siRNA 869, m_siRNA 150). mRNA level of endoglin in cells treated with siRNA molecules was normalized to the mRNA level of endoglin in cells treated with Lipofectamine RNAiMAX only (% of LF); *P<0.05 vs. untreated control cells (HMEC-1 or 2H11), LF and negative control siRNA (siRNA Ctrl). Data represent arithmetic mean±SEM. (C) Immunofluorescence staining of endoglin after the transfection of endothelial cells HMEC-1 with 3 different siRNA molecules (h_siRNA 529, h_siRNA 240, h_siRNA 241). (D) Immunofluorescence staining of endoglin after the transfection of endothelial cells 2H11 with 3 different siRNA molecules (m_siRNA 868, m_siRNA 869, m_siRNA 150). (E) Mean fluorescence intensity of HMEC cells stained for endoglin determined with flow cytometry analysis after the transfection of cells with 3 different siRNA molecules (h_siRNA 529, h_siRNA 240, h_siRNA 241). (F) Mean fluorescence intensity of 2H11 cells stained for endoglin determined with flow cytometry analysis after the transfection of cells with 3 different siRNA molecules (m_siRNA 868, m_siRNA 869, m_siRNA 150). Values are normalized to Lipofectamine RNAiMAX group (% of LF). *P<0.05 vs. untreated control cells (HMEC-1, 2H11), LF and siRNA Ctrl.
Figure 2.
Effect of siRNAs against endoglin on cell proliferation.
(A) Proliferation of HMEC-1 cells after the transfection with Lipofectamine RNAiMAX only (LF), h_siRNA 529, h_siRNA 240, h_siRNA 241 and negative control siRNA (siRNA Ctrl). (B) Proliferation of 2H11 cells after the transfection with Lipofectamine RNAiMAX only (LF), m_siRNA 868, m_siRNA 869, m_siRNA 150 and negative control siRNA (siRNA Ctrl). (C) Proliferation of TS/A cells after the transfection with Lipofectamine RNAiMAX only (LF), m_siRNA 868, m_siRNA 869, m_siRNA 150 and negative control siRNA (siRNA Ctrl). Proliferation of cells in each experimental group was normalized to day 0. *P<0.05 vs. untreated control cells (HMEC-1, 2H11 or TS/A), LF and siRNA Ctrl. Note that due to the slower growth of HMEC-1 cells, the y-axis has different range.
Figure 3.
Lipofection of HMEC-1 and 2H11 cells with siRNA against endoglin reduced tube formation.
Original images of tubular complexes of HMEC-1 cells (A) and corresponding binary masks (B) 2 days after the transfection with different siRNA molecules: untreated control cells (HMEC-1), cells transfected with Lipofectamine RNAiMAX only (LF), h_siRNA 529, h_siRNA 240, h_siRNA 241 and negative control siRNA (siRNA Ctrl). Original images of tubular complexes of 2H11 cells (C) and corresponding binary masks (D) 2 days after the transfection with different siRNA molecules: untreated control cells (2H11), cells transfected with Lipofectamine RNAiMAX only (LF), m_siRNA 868, m_siRNA 869, m_siRNA 150 and negative control siRNA (siRNA Ctrl). Scale bar: 100 µm.
Table 2.
Total length of tubular complexes, total size of tubular complexes and number of junctions after the transfection of endothelial cells HMEC-1 with 3 different siRNA molecules (h_siRNA 529, h_siRNA 240, h_siRNA 241).
Figure 4.
The effect of electrotransfer of siRNA against endoglin in TS/A tumors.
(A) Growth of TS/A tumors exposed to a single therapy: intratumoral injection of H2O alone (control) or combined with application of electric pulses (EP), intratumoral injection of m_siRNA 869, intratumoral injection of negative control siRNA (siRNA Ctrl) and electrotransfer of m_siRNA 869 (m_siRNA 869+EP) and negative control siRNA (siRNA Ctrl+EP). Arrow represents the day of treatment. (B) qRT-PCR analysis of endoglin mRNA level after a single therapy. (C) Growth of TS/A tumors exposed to repetitive therapies. Arrows represent the days of treatment. (D) qRT-PCR analysis of endoglin mRNA level after repetitive therapies. *P<0.05 vs. all groups.
Figure 5.
SiRNA against endoglin reduced the number of blood vessels, but not proliferation of tumor cells.
Immunohistologically stained tumor sections with anti-CD31 (A) and anti-Ki-67 (B) antibodies after repetitive treatments of TS/A tumors: intratumoral injection of H2O alone (control) or combined with application of electric pulses (EP), intratumoral injection of m_siRNA 869, intratumoral injection of negative control siRNA (siRNA Ctrl), electrotransfer of m_siRNA 869 (m_siRNA 869+EP) and negative control siRNA (siRNA Ctrl+EP). Scale bar: 50 μm. (C) Average number of blood vessels smaller than 30 μm per field of view in tumor sections at 30×magnification (D) Average number of Ki-67 positive cells per field of view in tumor sections at 30×magnification. *P<0.05 vs control, siRNA Ctrl and siRNA Ctrl+EP.
Table 3.
Sequences of siRNA duplexes used in the study.