Figure 6.

HEPES-Induced Protein Transfection Was Accomplished through Endocytosis and Charge Neutralization

(A–C) MDAH2774 cells were pretreated for 30 min with Opti-MEM (Ctrl), 0.4 M sucrose (an inhibitor of clathrin-mediated endocytosis), 5 μg/mL filipin (an inhibitor of caveolae-mediated endocytosis), or 0.25 mM amiloride (an inhibitor of macropinocytosis). Alexa Fluor 488-conjugated antibodies were delivered into cells using 20 mM HEPES, as described in The HEPES Method, followed by a 4-h incubation period. (A) Transfection efficiency was determined using a confocal fluorescent microscope. (B) Alexa Fluor 488 fluorescent intensity signals were quantified using Q-Win software and are expressed as relative ratios. (C) At 4 h of inhibitor treatment, cytotoxicity was analyzed using the LDH assay. Error bars indicate the SEM (n = 3).*p < 0.05. (D) To colocalize transfected proteins with the early endosome, we first used the HEPES method to transfect Alexa Fluor 546-conjugated antibodies into 786-O cells, and then we infected cells with CellLight Early Endosomes-GFP, BacMam 2.0 to express GFP-Rab5. To colocalize transfected proteins with the late endosome, we instead used Alexa Fluor 488-conjugated proteins and CellLight Late Endosomes-RFP, BacMam 2.0 to express RFP-Rab7. After 24 h of incubation, the live 786-O cells were examined using fluorescent microscopy. (E) Self-diffusion coefficients (Ds) were calculated at different molar ratios of STIP1 to HEPES (black squares). The Ds of the NMR internal standard (DSS) obtained under the same experimental conditions served as a reference (red circles). Ctrl, control; Ab, antibody; rh, recombinant human; DSS, dimethyl-silapentane-sulfonic acid.