nt.
Two important caveats were noted during the course of these experiments. RAW cells exhibited a greater tendency than HeLa cells to accumulate exogenous proteins on the cellular surface irrespective of incubation (Figure 6A) or electroporation (Figure 6B). Despite this, all electroporated samples had increased internal protein (Figures 1, 2, 5B). Additionally, the membrane-associated protein disappeared over time. A second caveat was a small population of cells exhibiting a phenotype consisting of smaller, rounded cells loaded with high amounts of exogenous proteins in both control (incubated) and treated (electroporated) cells (Figure 6 C and D, incubation samples shown). The nuclei of these cells showed condensed chromatin based on DAPI staining, and filled the entirety of the cellular volume, suggestive of apoptosis. It is therefore possible that apoptotic cells (arising as a normal part of the cell cycle or due to harvest/treatment) have a propensity to absorb protein from the buffer.
To demonstrate that electroporated proteins were functional and could localize correctly inside the host cell, co-localization and protein-protein interaction between the Salmonella effector protein SspH1 and its known host target, protein kinase N1 (PKN1) 33 was shown. After electroporation, the physical interaction between SspH1 and PKN1 was verified by an affinity based immunoprecipitation followed by western blot (Figure 7A). Co-localization was also observed by confocal microscopy, which indicates the fluorophores are close enough spatially to overlap 34. After electroporation with 50 µg/ml SspH1, HeLa cells (Figure 7B) were fixed and stained for SspH1 (green) and PKN1 (red). At low laser power distinct foci (yellow) were observed in the nucleus indicating SspH1 and PKN1 were physically close enough to interact. This interaction has been established in the literature; however this study is the first to show co-localization in the nucleus.
Figure 1: Electroporation of GFP - HeLa or RAW 264.7 cells. Cells were incubated or electroporated with 25 µg/ml of purified green fluorescent protein (GFP) and stained with anti-GFP antibody (Green), DAPI, a nuclear specific probe (Blue), and WGA (Red) to delineate the cytosolic boundary. Incubated (A) HeLa cells show an absence of green fluorescence indicating a lack of internalization of GFP. (B) shows a representative photomicrograph of electroporated GFP. Note the appearance of intracellular foci indicating GFP had entered the cells. (C) and (D) are representative images of RAW cells incubated (C), or electroporated (D) with 25 µg/ml of purified green fluorescent protein.
Figure 2: Electroporation of Salmonella effector GtgE - HeLa cells. Cells were incubated (A) or electroporated (B) with 50 µg/ml of an affinity tagged Salmonella effector, GtgE, and stained with an anti-effector tag antibody (Green), DAPI, a nuclear mask (Blue), and WGA (Red) to delineate the cytosolic boundary. In (A), incubated HeLa cells show an absence of green fluorescence indicating a lack of internalization of GtgE. (B) shows a representative photomicrograph of electroporated GtgE, showing intracellular electroporated effector protein. (C) and (D) are representative images of RAW cells that were either incubated or electroporated, respectively, in the same fashion with 50 µg/ml of Salmonella GtgE. Light blue is the combination, or overlay, of red fluorescence from the WGA and green fluorescence from the secondary antibody.
Figure 3: Titration of electroporated protein – RAW macrophage-like cells. Cells were electroporated with 2.5 μg/ml (A), 25 μg/ml (B), or 50 μg/ml (C) Salmonella effector GtgE and allowed to recover for 4 hr. The cells were fixed and stained with an anti-SBP-tag antibody (green) and the nuclei mask, DAPI (blue). It is possible to visualize fluorescent foci, indicative of intracellular GtgE at 2.5 μg/ml via confocal microcopy, although better results were obtained when higher starting amount of protein were used. Satisfactory results (including intracellular protein easily observed by confocal mic