Dextran (150?kDa) conjugated to FITC was purchased from TdB. the listeriolysin O (LLO)-mediated resealing technique, 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 Enzaplatovir (8-OH-cAMP) or specialty peptide (Akt-in), and exhibited PKA activation or Akt inhibition, respectively. Collectively, the LLO-type resealing method is usually a user-friendly and reproducible intracellular delivery system for mid-sized membrane-impermeable molecules into cells and for evaluating their intracellular activities. Introduction Cell-based assays have become increasingly important in preclinical studies for drug discovery. Such assays enable the detailed study of the mechanisms of drug action, speeding up development time and reducing costs. Recently, biopharmaceutical products such as nucleotides, peptides, and antibodies have received increased attention owing to their higher substrate specificities and are thought to overcome certain disadvantages of small-molecule compounds1C3. In Enzaplatovir particular, mid-sized peptides (less than ~10?kDa) can be chemically synthesized, unlike antibodies, and are expected to reduce the cost in development and production of drugs. One example is usually CP2, a cyclic peptide inhibitor of histone demethyrase4, which is a modified, cyclic compound comprising natural and unnatural amino acids. However, for intracellular targets, very high concentrations of proteins and cytoskeletal or membranous structures in the cells might affect the activity that was measured in the system5C7, which is a crucial issue Enzaplatovir for drug efficacy and design. Additionally, such mid-size products are generally membrane impermeable and methods to introduce them into cells have also been extensively studied8,9. Thus, to test their efficacy, these products should be introduced into cells across the plasma membranes and their activity should be evaluated in cells. Several methods for introducing molecules into cells have been developed: microinjection10,11, electroporation12, cell-penetrating peptides (CPPs)13. There are both advantages and disadvantages to each method. Microinjection can be performed using commercially available gear, but may be difficult to apply to high-content analyses. Recent advances in electroporation enable delivery of various types of molecules such as proteins, nucleotides, and small chemical compounds into cells using dedicated equipment, but it is usually inadequate for large-scale studies and can cause damage to cells. CPPs are peptides of typically 5C30 amino acids that can facilitate uptake of linked cargo into cells. CPP-based delivery of molecules into cells is usually less toxic, allowing its therapeutic use, but CPP conjugation to cargo molecules is required, which might perturb the cargos function. We previously described a cell-resealing technique that makes use of the temperature-dependent pore-forming activity of the streptococcal toxin, streptolysin O (SLO), to introduce various molecules into cells14. SLO is usually a cholesterol-dependent cytolysin (CDC) Enzaplatovir derived from functional analysis of membrane-impermeable low-molecular weight molecule by LLO-type resealing One of the aims of this study is usually to evaluate the intracellular activity of delivered biomolecule in resealed cells. We next examined the intracellular activation of protein kinase A (PKA) by cAMP or its membrane-impermeable/permeable analogues. We first investigated the phosphorylation of PKA substrate protein by the membrane permeable cAMP analogue, db-cAMP, to find suitable substrate proteins that could serve as a sensitive indicator for PKA activation. HeLa cells were treated with db-cAMP (Mw?=?491.4) or H89, a membrane permeable inhibitor of PKA, at varying concentrations for 60?min. The cells were lysed and subjected Enzaplatovir to Western blotting using anti-phospho- (Ser/Thr) PKA substrate antibody. As shown in Fig.?S7, we detected nine polypeptide bands that were phosphorylated in the presence of db-cAMP but not of H89. Band e, one of the polypeptide bands that responded to db-cAMP treatment as above, was chosen as a sensitive indicator for quantitative PKA activation, although we were unable to identify this polypeptide band. Next, using the same experimental procedure, we examined the effect of the membrane impermeable cAMP analogue, 8-OH-cAMP (MW?=?367.2)27, on PKA activation in LLO-type resealed cells. LLO-mediated permeabilized HeLa cells were Rabbit Polyclonal to HCFC1 incubated with 1?mM 8-OH-cAMP or 1?mM db-cAMP for 30?min and resealed. Then, the cells were incubated for another 60?min with medium and lysed. WB analysis revealed that this intensity of band e was significantly increased by 8-OH-cAMP, indicating that introduction of 8-OH-cAMP into LLO-type resealed HeLa cells successfully activated PKA (Fig.?6A,B). We also confirmed that this activation was dependent upon LLO (Fig.?6C,D). Interestingly, the membrane permeable cAMP analogue, db-cAMP, activated PKA to a lesser extent than 8-OH-cAMP (Fig.?6A,B). We suppose that intracellularily delivered db-cAMP might diffuse from the cells through the plasma membrane during incubation owing to its membrane permeability. As such, LLO-type resealing might show useful for evaluating the intracellular retention of various introduced compounds, which could be beneficial for understanding the efficacy and the side effect of drugs. Furthermore, we performed the comparable experiments using murine lymphoma EL4 cells, confirming that this method was also applicable for suspended cell systems (Fig.?S8). Open in a separate window Physique 6 Activation of PKA by.