LLO-mediated Cell Resealing System for Analyzing Intracellular Activity of Membrane-impermeable Biopharmaceuticals of Mid-sized Molecular Weight

Cell-based assays have become increasingly important in the preclinical studies for biopharmaceutical products such as specialty peptides, which are of interest owing to their high substrate specificity. However, many of the latter are membrane impermeable and must be physically introduced into cells to evaluate their intracellular activities. We previously developed a “cell-resealing technique” that exploited the temperature-dependent pore-forming activity of the streptococcal toxin, streptolysin O (SLO), that enabled us to introduce various molecules into cells for evaluation of their intracellular activities. In this study, we report a new cell resealing method, the listeriolysin O (LLO)-mediated resealing method, to deliver mid-sized, membrane-impermeable biopharmaceuticals into cells. We found that LLO-type resealing required no exogenous cytosol to repair the injured cell membrane and allowed the specific entry of mid-sized molecules into cells. We use this method to introduce either a membrane-impermeable, small compound (8-OH-cAMP) or specialty peptide (Akt-in), and demonstrated PKA activation or Akt inhibition, respectively. Collectively, the LLO-type resealing method is a user-friendly and reproducible intracellular delivery system for mid-sized membrane-impermeable molecules into cells and for evaluating their intracellular activities.


Figure. S1 LLO-type resealing of MEF and L cells.
Mouse embryonic fibroblasts (MEF) or mouse methylcholanthrene-induced sarcoma cells (L-cell) were incubated with 0.23 or 0.11 µg/ml LLO respectively, and were incubated for 10 min at 37°C in TB that contained propidium iodide. The cells were incubated with resealing buffer that contained 10 kDa dextran conjugated with fluorescein at 37°C for 30 min. After incubation with 1 mM CaCl 2 for 5 min, the cells were incubated with medium at 37˚C for 1 h and observed using confocal microscopy. Bar = 50 µm. Semi-intact HeLa cells, that were stained with propidium idodide, were incubated with L5178Y cytosol, an ATP regeneration system, GTP and glucose (Cytosol and ATP R.S/GTP/Glu), ATP regeneration system, GTP, and glucose (Without cytosol), or L5178Y cytosol (Without ATP R.S/GTP/Glu) at 37˚C for 30 min. The cells were fixed with 4 % paraformaldehyde in PBS for 20 min, and then permeabilized with 0.2 % Triton X-100 in PBS for 15 min at room temperature. After blocking with 3 % BSA for 30 min, the cells were stained with mouse anti-β Tubulin antibody and DAPI (5 µg/ml). The cells were observed using confocal microscopy. Bar = 100 µm.

Fig. S4 Recycling of transferrin in LLO resealed cells.
HeLa cells were permeabilized with 0.15 µg/ml LLO. After resealing with 1 mM CaCl 2 , the cells were incubated with medium for 4 h. After washing with PBS twice, the cells were incubated with 10 µg/ml alexa 488 conjugated human transferrin (molecular probes) in serum free medium for 30 min at 37℃ to allow uptake and delivery to endosomal compartments, and then chased in medium contained excess non-labeled transferrin (Roche) in the presence or absence of 10 µM monensin (SIGMA) for various periods of time. The cells were washed twice with PBS, and were observed by using a confocal microscope or were subjected to flow cytometry.  HeLa cells were permeabilized with 0.15 µg/ml LLO and incubated with resealing buffer containing ATP regeneration system, GTP and glucose for 30 min. After resealing with 1 mM CaCl 2 , the cells were incubated with medium at 37℃ 5% CO 2 for overnight. Then, intact or resealed HeLa cells were seeded at 3×10 4 cell/well at 24 well plate, and incubated with medium for 1, 2 or 3 days. The cells were observed by bright field microscopy. Bar = 200 µm. HeLa cells were incubated with or without 0.15 µg/ml LLO on ice for 5 min. After washing with TB three times, the cells were incubated in preheated TB at 37℃ for 10 min. After lysing the cells, total protein was separated using SDS-PAGE and subjected to CBB staining. The number of cells per well is indicated at the top of the figure. No significant difference in the protein band pattern was observed, indicating that the composition of the proteins appeared similar.

Figure. S7 Band e is a sensitive PKA substrate read-out of cAMP stimulation.
HeLa cells were incubated with DMSO, 0.3, 3, 30 or 60 µM H89 or 10, 100, 1000, 2000 µM db-cAMP in medium at 37℃ for 60 min. The cells were lysed, and were subjected to Western blotting using antibodies against Phospho-PKA substrate and GAPDH. The arrows (band-a to -i) indicate the bands, the intensity of which were increased in dependence on the db-cAMP concentration. Band e is the band that increased its intensity the most following db-cAMP treatment. The relative intensity of band e to GAPDH is shown in the right graph.

LLO-type resealed EL4 cells.
(A, B) 2×10 6 of EL4 cells were suspended in RPMI medium without serum and centrifuged at 300 g for 5 min at 4˚C. The pelleted cells were resuspended in 0.15 µg/ml LLO in RPMI and rotated at 4˚C for 12 min. After washing the cells by centrifugation and the subsequent suspension of the cell pellet in RPMI medium without serum three times, the cells were resuspended and incubated with pre-warmed TB at 37˚C for 10 min to permeabilize the cell membrane. The cells were centrifuged and the cell pellet was suspended in TB, and the washing process was repeated once.
The pellet of permeabilized EL4 cells were suspended and incubated with resealing buffer that contained 3 kDa fluorescein-dextran in the presence or absence of 1.5 mg/ml L5178Y cytosol at 37˚C for 30 min. After the addition of CaCl 2 to the final concentration of 1 mM and the further incubation at 37˚C for 5 min to repair the lesion of cell membrane, RPMI medium was added and centrifuged at 300 g for 5 min. The resealed EL4 cells were washed with medium by centrifugation and the subsequent resuspension with medium once, and were incuabated at 37˚C 5 % CO 2 for 30 min. indicate the band that the intensity of which were increased in dependence on the db-cAMP concentration. Band 4 is the band that increased its intensity the most following db-cAMP treatment. The relative intensity of band e to β-Tubulin is shown in the right graph. (D) After permeabilizing EL4 cells as described (A), the semi-intact EL4 cells were incubated with resealing buffer with or without 1 mM 8-OH-cAMP, which is a membrane-permeable cAMP analogue, at 37˚C for 30 min. the cells were resealed by addition of 1 mM CaCl 2 at 37˚C for 5 min, and further incubated with medium at 37˚C at 5 % for 1 h. The cells were lysed and were subjected to Western