87 ± 1.82) (Figure 3A). In conclusion, selleck screening library Ad-bFGF-siRNA inhibits IL-6 cytokine expression in a time-dependent manner. Figure 3 Ad-bFGF-siRNA reduces IL-6 secretion in U251 cells. (A) ELISA analysis showed that IL-6 secretion in the Ad-bFGF-siRNA group (MOI = 100) was lower than that in the control and Ad-GFP
groups during both 24-48 h and 48-72 h periods. **: p < 0.0001. Data are presented as mean ± SD, n = 3. (B) U251 cells infected with Ad-bFGF-siRNA for 48 h were treated with serum-free DMEM in the presence or absence of recombinant IL-6 (100 ng/ml) for 24 h. Cells treated with DMSO for 72 h served as controls. Erismodegib supplier The phosphorylation of STAT3 at both Tyr705 and Ser727 is elevated after stimulated with IL-6 for 24 h. To explore whether exogenous IL-6 can rescue Ad-bFGF-siRNA-inhibited STAT3 activation, U251 cells infected for 48 h were treated with serum-free DMEM in check details the presence or absence of recombinant
IL-6 (100 ng/ml) for 24 h. Cells treated with DMSO for 72 h were used as a negative control. As shown in Figure 3B, the phosphorylation of STAT3 at both Tyr705 and Ser727 was elevated after stimulated with IL-6 for 24 h. 3.4 Ad-bFGF-siRNA induces depolarization of mitochondria and apoptosis in U251 cells Given the central role of mitochondria in orchestrating the apoptotic processes, we assessed the mitochondrial transmembrane potential (ΔΨm) after bFGF knockdown by Ad-bFGF-siRNA using JC-1 staining. JC-1 forms high orange-red fluorescent J-aggregates (FL-2
channel) at hyperpolarized membrane potentials and weak green fluorescent monomers (FL-1 channel) at depolarized membrane potentials. The results showed that the control and Ad-Null cells exhibited high orange-red fluorescence and weak green fluorescence (Figure 4A), indicating hyperpolarized mitochondria. In contrast, after treated Tangeritin with Ad-bFGF-siRNA (MOI = 100) for 72 h, an increased subpopulation of cells displayed decreased orange-red fluorescence, suggesting the collapse of mitochondrial membrane potentials. The ratio of cells with high membrane potentials in the Ad-bFGF-siRNA group (90.87 ± 1.84%) decreased significantly from that in the control and Ad-Null groups (92.12 ± 2.50% and 74.42 ± 4.66%, respectively; p < 0.0005) Figure 4 Ad-bFGF-siRNA reduces the mitochondrial transmembrane potential (ΔΨm) and induces apoptosis in U251 cells. (A) Cytofluorimetric analysis using JC-1 staining demonstrated that Ad-bFGF-siRNA treatment (MOI = 100) induces depolarization of mitochondria. Percentages of cells with high ΔΨm (%) are shown in each column. Data are represented as mean ± SD of three replicates (**: P < 0.0005). Changes in ΔΨm were also detected by fluorescence microscopy. Magnification: 200×. Scale bar: 50 μm. Normal cells that have high ΔΨm show punctuate yellow fluorescence. Apoptotic cells show diffuse green fluorescence because of the decrease in mitochondrial membrane potential.
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