Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells

Figure 1 Identification of recombinant vectors. BamHI and PstI were used to digest and identify the recombinant plasmids. The results show that all plasmids were positive for the recombinant vector; M: Marker; 1: p-OPN-siRNA-1; 2: p-OPN-siRNA-2; 3: p-OPN-siRNA-3; 4: p-OPN-siRNA-4; 5: p-NC-siRNA.

Figure 2 Verification of the recombinant vectors. Sequencing of the recombinant vectors (performed by Sangon Co.) showed that all coding sequences were inserted correctly with no mutations; A: p-OPN-siRNA-1; B: p-OPN-siRNA-2; C: p-OPN-siRNA-3; D: p-OPN-siRNA-4.

Figure 3 Confirmation of osteopontin knockdown by real-time reverse transcription-polymerase chain reaction. The expression of OPN mRNA was significantly decreased in Lovo cells by p-OPN-siRNA-1, -2, -3, and -4 (^aP < 0.05 vs Lovo), with the largest reduction observed with p-OPN-siRNA-4 (^cP < 0.05 vs Lovo-OPN-4). OPN: Osteopontin.

Figure 4 Confirmation of osteopontin knockdown by western blot. The expression of OPN protein was significantly decreased in Lovo cells by p-OPN-siRNA-1, -2, -3, and -4 (^aP < 0.05 vs Lovo), with the largest reduction observed with p-OPN-siRNA-4 (^cP < 0.05 vs Lovo-OPN-4). OPN: Osteopontin.

Figure 5 Cell adhesion assay. Staining with 0.1% crystal violet (magnification × 100) revealed reduced adhesion in cells expressing Lovo-OPN-4 (A-C); Quantification of adhesion, obtained by absorbance at 595 nm (D) (n = 15; ^aP < 0.05 vs Lovo-OPN-4; ^cP < 0.05 vs Lovo). OPN: Osteopontin.

Figure 6 Cell invasion assay. Staining with 0.1% crystal violet (magnification × 200) revealed a reduced number of invasive Lovo-OPN-4 cells (A-C); Quantification of invasion (D) (n = 15; ^aP < 0.05 vs Lovo-OPN-4; ^cP < 0.05 vs Lovo). OPN: Osteopontin.