T they also overexpress VEGF protein in hypoxia conditions [47,48]. A 15 mM modified SL2-B concentration was used in this study but our results showed that both MCF-7 and HCT-116 cancer cells displayed only 2363.2 and 961.8 decrease in cell proliferation was observed respectively. Based on these cell proliferation results, the effect of PS-modified SL2-B sequence on cell proliferation is believed to be cell type specific. Since antiproliferative effect on MCF-7 and HCT-116 cancer cells were not very substantial, they were not used for further studies below. Additional antiproliferative studies on various cancer cell types should be conducted to uncover the potential therapeutic targets and to identify the factors responsible for cell specific antiproliferative activity of this aptamer.Flow Cytometry and Western Blot Analysis of JRF 12 web Jagged-1 Protein ExpressionNotch signaling is an evolutionary conserved signaling pathway affecting many cellular processes such as cell-fate determination, differentiation, proliferation, and survival. Five Notch ligands (Jagged-1, Jagged-2, Delta-1, Delta-3, and Delta-4) and four Notch receptors have been well established in mammals [49,50]. Evidence indicates the biochemical linkage between VEGF and delta/jagged-notch pathways activation, and together both are involved in promoting tumor progression [51,52]. In this linkage, VEGF pathway is essential for the initiation of tumor Danusertib chemical information angiogenesis and acts as the upstream activating stimulus, whereas notch signaling which acts on downstream of the VEGF pathway, helps to respond to activating stimulus and shape the activation by making cell fate decisions [49]. Due to the crosstalk between VEGF and notch signaling pathways, the effect of PS-modified SL2-B aptamer was tested on Jagged-1, which is one of the notch ligands. Jagged-1 is overexpressed in various malignant tumors and has been associated with 1531364 cancer recurrence [53?5]. Here, we examined the effect of PS-modified SL2-B aptamer on the expression of Jagged-1 protein in Hep G2 cells via flow cytometry technique. Compared to the untreated sample (only cells), modified SL2-B treatment exhibited decrease in the fluorescent signal (Figure 8). This shift in the peak indicates the downregulation of the Jagged-1 expression due to the addition of PSmodified SL2-B aptamer in Hep G2 cells (p-value ,0.05). Besides flow cytometry, the effect of PS-modified SL2 aptamer on Jagged-1 protein expression in Hep G2 cells was analyzed using western blotting. The scrambled sequence of the modified aptamer was used as control. The modified aptamer appears to induce a lower expression of the Jagged-1 protein in Hep G2 cells as compared to the scrambled sequence (Figure 9). This confirms the sequence specific inhibition of the aptamer on Jagged-1 protein expression in Hep G2 cells. Based on both flow cytometry and western blotting results, it can be concluded that the binding of PSmodified SL2-B aptamer to VEGF protein exhibits its antiprolifdownstream VEGF linked intracellular signaling pathways. The result also indicates that VEGF protein may be involved in the proliferation of investigated Hep G2 cancer cells under hypoxia conditions. On the contrary, the unmodified SL2-B aptamer sequence did not exhibit significant inhibitory activity on the cellular proliferation. This could be due to the degradation of the unmodified sequence by nuclease enzymes in the media before pronouncing its effect on the cancer cells. To demonstrate that.T they also overexpress VEGF protein in hypoxia conditions [47,48]. A 15 mM modified SL2-B concentration was used in this study but our results showed that both MCF-7 and HCT-116 cancer cells displayed only 2363.2 and 961.8 decrease in cell proliferation was observed respectively. Based on these cell proliferation results, the effect of PS-modified SL2-B sequence on cell proliferation is believed to be cell type specific. Since antiproliferative effect on MCF-7 and HCT-116 cancer cells were not very substantial, they were not used for further studies below. Additional antiproliferative studies on various cancer cell types should be conducted to uncover the potential therapeutic targets and to identify the factors responsible for cell specific antiproliferative activity of this aptamer.Flow Cytometry and Western Blot Analysis of Jagged-1 Protein ExpressionNotch signaling is an evolutionary conserved signaling pathway affecting many cellular processes such as cell-fate determination, differentiation, proliferation, and survival. Five Notch ligands (Jagged-1, Jagged-2, Delta-1, Delta-3, and Delta-4) and four Notch receptors have been well established in mammals [49,50]. Evidence indicates the biochemical linkage between VEGF and delta/jagged-notch pathways activation, and together both are involved in promoting tumor progression [51,52]. In this linkage, VEGF pathway is essential for the initiation of tumor angiogenesis and acts as the upstream activating stimulus, whereas notch signaling which acts on downstream of the VEGF pathway, helps to respond to activating stimulus and shape the activation by making cell fate decisions [49]. Due to the crosstalk between VEGF and notch signaling pathways, the effect of PS-modified SL2-B aptamer was tested on Jagged-1, which is one of the notch ligands. Jagged-1 is overexpressed in various malignant tumors and has been associated with 1531364 cancer recurrence [53?5]. Here, we examined the effect of PS-modified SL2-B aptamer on the expression of Jagged-1 protein in Hep G2 cells via flow cytometry technique. Compared to the untreated sample (only cells), modified SL2-B treatment exhibited decrease in the fluorescent signal (Figure 8). This shift in the peak indicates the downregulation of the Jagged-1 expression due to the addition of PSmodified SL2-B aptamer in Hep G2 cells (p-value ,0.05). Besides flow cytometry, the effect of PS-modified SL2 aptamer on Jagged-1 protein expression in Hep G2 cells was analyzed using western blotting. The scrambled sequence of the modified aptamer was used as control. The modified aptamer appears to induce a lower expression of the Jagged-1 protein in Hep G2 cells as compared to the scrambled sequence (Figure 9). This confirms the sequence specific inhibition of the aptamer on Jagged-1 protein expression in Hep G2 cells. Based on both flow cytometry and western blotting results, it can be concluded that the binding of PSmodified SL2-B aptamer to VEGF protein exhibits its antiprolifdownstream VEGF linked intracellular signaling pathways. The result also indicates that VEGF protein may be involved in the proliferation of investigated Hep G2 cancer cells under hypoxia conditions. On the contrary, the unmodified SL2-B aptamer sequence did not exhibit significant inhibitory activity on the cellular proliferation. This could be due to the degradation of the unmodified sequence by nuclease enzymes in the media before pronouncing its effect on the cancer cells. To demonstrate that.
