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. 2021 Apr 14;13(4):552. doi: 10.3390/pharmaceutics13040552

Figure 1.

Figure 1

The optimization of TP internalization and induced bystander uptake. (A) Quantification of TP peptide uptake in various cell lines. Cells were treated with 10 μM of FAM-labeled TP peptide (FAM-TP) at 37 °C for 1 h. After washing, the fluorescence intensity of TP peptide per sample was detected and measured by flow cytometry. The median fluorescence value was normalized to that of corresponding cells alone (y-axis). Error bars, mean ± standard deviation (s.d.) (n = 3). Statistically significant difference between various cell lines was determined by one-way ANOVA. F (15, 32) = 180.5, p < 0.0001. The uptake in CHO-pgs745 was significantly lower in comparison with that in CHO-K1 when two tailed Student’s t-test was performed (p < 0.0001). (B) Quantification of TP and bystander AgNP uptake by CHO cells at different concentrations of TP. As described in the Methods, CHO cells were incubated with AgNP and TP peptide together at the indicated concentrations (5, 10, 20, 50 μM). After washing, the fluorescence intensity per sample was quantified by flow cytometry. The median fluorescence intensity of internalized bystander AgNP was normalized to that of cells incubated with AgNP alone (left y-axis, black). Error bars, mean ± standard deviation (s.d.) (n = 3). Relative AgNP uptake was not significantly different from each other using one-way ANOVA with Tukey’s multiple comparisons test. Left y-axis, relative AgNP uptake (black). Right y-axis, relative TP uptake (red). (C) Confocal images of CHO cells after incubation with FAM-TP (green) and bystander AgNP (AgNP-555, red). Representative images were shown here. Scale bars, 10 μm. (D) Quantification of bystander AgNP uptake by CHO cells. Mean fluorescence intensity of AgNP-555 obtained from three independent experiments (n = 3) through Image J software was normalized to that of AgNP alone group (y-axis). Values shown here were mean ± s.d. There was a statistically significant difference between AgNP alone group and TP group as determined by two tailed Student’s t-test. **** p < 0.0001.