Fig 1: Analysis of siRNA/HE25 complex receptor binding, SARS-CoV-2 cellular receptor inhibition, and endosomal release(A) Binding of HE25 to ACE2, ITGAV, NRP1, and ITGA5 as analyzed by ELISA. ITGB3, ITGA2, ITGA2B, and FIB were included as negative controls. (B) Receptors binding to SARS-CoV-2 spike RBD. (C) ELISA showing HE25 binding to SARS-CoV-2 spike RBD. (D) Competitive ELISA showing the inhibition of spike RBD binding to ACE2 and ITGAV receptors by increasing concentrations of HE25. ITGA2 receptor was used as a negative control. (E) EMSA showing the release of the siRNA from the complex in the condition of late endosome. Lanes 1 and 7 contain ladder; lanes 2 and 3 contain free siRNA and siRNA-peptide complex at physiological pH 7.4; lanes 4 and 5 contain siRNA and siRNA-peptide complex at late endosomal pH 5.5; and lane 6 contains siRNA-peptide complex at late endosomal pH 5.5 in the presence of cathepsin B, release of siRNA can be observed. (F) Evaluation of GAPDH silencing in Vero E6 cells using GAPDH siRNA/HE25 complex at various molar ratios, in the presence and absence of 10 nM CA-074-ME, a cathepsin B inhibitor. Error bars indicate mean ± SD based on three biological replicates. Data were analyzed using GraphPad Prism, employing a two-way ANOVA and multiple comparison tests (∗p < 0.0332; ∗∗p < 0.0021; ∗∗∗p < 0.0002; ∗∗∗∗p < 0.0001) for statistical significance evaluation.
Fig 2: Internalization mechanisms of siRNA/HE25 in Vero E6 cells(A) Assessment of GAPDH silencing in Vero E6 cells by GAPDH siRNA/HE25 complex across various molar ratios, using different internalization pathway inhibitors. (B) ImageJ quantitative analysis of colocalization from confocal microscopy images at diverse molar ratios. (C) Analysis of colocalization between Alexa Fluor 555-tagged siRNA/HE25 complex and Rab 4, Rab 9, Rab11A (markers for early, late, and recycling endosomes) or Integrin Alpha V (ITGAV). Selected cells are shown at a magnification of 5× from a representative biological replicate at a 1:40 M ratio. Non-magnified images are available in Figure S3. Rab4, Rab9, Rab11A, or ITGAV are shown in green pseudocolor (first column panels); Alexa Fluor 555 siRNA/HE25 in magenta pseudocolor (second column panels); and HSC Cell Mask Deep Red representing cell nuclei and cytoplasm in blue pseudocolor (third column panels). Areas of colocalization between green and magenta are depicted in white, signifying the spatial interaction between the siRNA/HE25 complex and endosomal or ITGAV markers. Pseudocolors were selected to enhance accessibility for individuals with color vision deficiencies. The scale bar in the upper right corner of the composite panels represents 5 μm. Error bars indicate mean ± SD based on three biological replicates. Data were analyzed using GraphPad Prism, employing a two-way ANOVA and multiple comparison tests (∗p < 0.0332; ∗∗p < 0.0021; ∗∗∗p < 0.0002; ∗∗∗∗p < 0.0001) for statistical significance evaluation.
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