FIG. 9.
Endocytosis inhibition and tracking studies. (A) Inhibition experiments. iNOS-induced RAW 264.7 cells were incubated with cytochalasin D (Cyto-D) (average of 2–50 μM), chlorpromazine (CPZ) (20 μM) and methyl-beta-cyclodextrin, (MCD) (20 μM) for 1 hour and then incubated with PAEA160-b-PS30 AlexaFluor488-cSCKpa100 (10 μg/mL) for another hour, and cSCK uptake (n=3) was determined by flow cytometry. (B) Degree of non-colocalization of siRNAs and cSCKs with Lysotracker in the presence and absence of GALA. Induced RAW264.7 cells were incubated with siRNA (50 nM)•AlexaFluor488-cSCKpa100 (10 μg/mL)-Cy5•GALA (0, 0.2 μM) complexes for 22 hours, after which Lysotracker-Blue (50 nM) was added. Two hours later the cells were analyzed by confocal microscopic imaging. The degree of non-colocalization of siRNA with cSCK (siRNA/cSCK), siRNA with Lysotracker (siRNA/Lyso), cSCK with Lysotracker (cSCK/Lyso), and Lysotracker with siRNA and cSCK (Lyso) was determined by Image J Green and Red Puncta Colocalization Macro (n=7 images without GALA, and n=10 with GALA with 2–3 cells/image). (C) Degree of non-colocalization of siRNAs and cSCKs with dextran-cascade blue. AlexaFluor488-cSCK-pa100 (10 μg/mL), Cy5-siRNA (50 nM), and GALA (0, 0.2 μM) with dextran-cascade blue (100 μg/mL) were transfected to RAW264.7 cells for 8 hours followed by confocal microscopic imaging. The degree of non-colocalization of siRNA with cSCK (siRNA/cSCK), siRNA with dextran (siRNA/Dex), cSCK with dextran (cSCK/Dex) and dextran with siRNA and cSCK (Dex) was determined as above (n=10 images without GALA and n=16 with GALA with 2–3 cells/image).