Protein transduction and confocal microscopy

Protein transduction was carried out as described previously with indicated modifications. Briefly, 4 × 10⁵ HeLa cells were plated onto glass-bottom dishes (Mattek) and on the next day cells were washed thrice with DMEM (Gibco). Flashed frozen recombinant proteins were thawed and briefly centrifuged at 20,000g on table top centrifuge at 4 °C. Cells were treated with recombinant proteins (25, 75 and 100 nM) in 800 μl of DMEM; after 4–5 h DMEM with 20% FCS containing penicillin (100 μg ml⁻¹) and streptomycin (100 μg ml⁻¹) was added to cells and they were incubated overnight. Next, live and dead cells were quantified in the EGFP-positive cell populations. Cellular uptake of recombinant proteins was monitored using confocal imaging microscopy. Cells were treated with 100 nM of recombinant proteins for 4 h, cells were washed with PBS, fixed with 4% formaldehyde. The cells were imaged using an Olympus Fluoview 1000 confocal laser scanning microscopy with a × 60/1.3 NA silicon oil lens. Images were acquired with a z-step size of 0.2 μm, a pinhole of 0.9 AU and line averaging of 48. The images were analysed using Fiji software.