 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 microscopy without excessive membrane associated aggregates) were obtained at 50 μg/ml for GtgE and thus no greater concentrations were tested.
Figure 4: Protein internalization and lack of co-localization with endosome markers. Consecutive optical slices (z-stacks) obtained by confocal microscopy to visualize if the electroporated protein was internal. RAW cells were electroporated with 50 µg/ml GtgE. (A) shows optical slice 6 of 36, the z-stack (2.1 micrometers) above the bottom of the dish. (B) shows the same field of view but at slice 26 of 36. The intracellular foci extend throughout the cell indicating that the protein is truly intracellular and not aggregated on the surface of the cell. Light blue is the combination of red WGA, green secondary antibody, and blue DAPI. Co-localization assay with markers of endosomes/lysosomes, Rab5, (C) or a lysosome marker, LAMP1 (D). The effector protein has been stained green, the markers (Rab5 or LAMP1) stained red, and the nuclei delineated with DAPI, blue. There is no evidence of co-localization with either LAMP1 or Rab5 indicating that the protein was not phagocytized.
Figure 5: Protein persistence after electroporation. Confocal micrograph showing the persistence of electroporated GtgE over time. 50 µg/ml GtgE was electroporated into HeLa cells. The cells were then fixed and stained with an anti-SBP-tag antibody (green) and the nuclei mask, DAPI (blue). 4 hr (A) was determined to be the minimum amount of time to allow cells to recover and as expected shows maximum intracellular protein. After 24 hr (B) and 96 hr (C), green foci, both intracellular and on the cellular surface, show effector persistence indicating that the protein is not degraded days after the initial treatment. The light blue color in this image is the overlay of blue DAPI and the green antibody staining.
Figure 6: Cell surface protein aggregation and an electroporation phenotype. RAW macrophage-like cells were either incubated (A) or electroporated (B) with 50 µg/ml GtgE and stained with anti-SBP-tag 