293 cells were transiently transfected with a DNA plasmid containing the same combination of luciferase reporter gene and CMV promoter present in the genome of our bacteriophage constructs. Cells were transiently transfected with this plasmid DNA using Lipofectamine, and were maintained in the presence or absence of bortezomib (10 nM) for 16 hours. Cells were then washed, and returned to normal medium. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to bortezomib had no effect on plasmid-mediated luciferase gene expression.

Lambda phage particles likely enter mammalian cells via a non-specific macropinocytotic uptake mechanism (Lankes et al., 2007) (A), and enter early endosomes (B). The phage-containing endosomes then either (i) undergo acidification and fusion with lysosomes, resulting in the degradation of phage particles by lysosomal proteases (C) or (ii) undergo lysis or recycling (Ding et al., 2006) (D), resulting in entry of the phage particles into the cytoplasm (E). Inhibition of lysosomal proteases results in stabilization of phage within lysosomes, allowing some phage particles to escape from this compartment and enter the cytoplasm (denoted by the dotted arrow; F). The majority of lambda phage particles within the cytosol undergo proteasomally-mediated degradation (G), but a fraction of the particles successfully uncoat and deliver their genomic payload to the nucleus, resulting in the expression of phage-encoded genes (i.e., the luciferase reporter gene in our vector constructs) (H).

Luciferase encoding phage particles displaying either a wild-type gpD protein (WT), a Tpell motif (Tpell) or a Tpell motif containing a mutated, non-functional PEST consensus element (Tpell-SA) were generated, and used to transduce 293 cells. Cells were exposed to luciferase-encoding phage particles at a MOI of 1×10⁶. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to the Tpell-SA phage resulted in an enhanced efficiency of phage-mediated luciferase gene expression, when compared to Tpell phage (the asterisk denotes p < 0.05, when compared to cells exposed to Tpell phage, as determined by two tailed paired t test). The results for WT phage in this Figure derive from data shown in Figure 1; these data were included here to facilitate comparison of the efficiency of phage-mediated gene transfer between Tpell-SA phage, Tpell phage and unmodified WT phage.

(A) 293 or COS cells were incubated with luciferase-encoding Tpell phage at a MOI of 1×10⁶. The indicated endosomotropic drugs were added to the culture medium at doses of 50 μM (omeprazole), 500 ng/ml (brefeldin A) and 50 μM or 70μM (chloroquine) for 293 and COS cells, respectively. 24 hours later the cells were washed, and placed in medium lacking the endosomotropic drugs. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to omeprazol or brefeldin A had no effect on phage-mediated luciferase gene expression. In contrast, treatment of cells with high concentrations of chloroquine resulted in a statistically significant enhancement of phage-mediated gene transfer (the asterisk denotes p < 0.05, when compared to untreated cells, as determined by one-way ANOVA with Tukey’s post-test). (B) 293 cells were incubated with luciferase-encoding Tpell phage at a MOI of 1×10⁶. Chloroquine was added to the culture medium at the indicated doses. 24 hours later the cells were washed, and placed in medium lacking the drug. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to a high dose of chloroquine resulted in a statistically significant enhancement of phage-mediated gene transfer (the asterisk denotes p < 0.05, when compared to untreated cells, as determined by one-way ANOVA with Tukey’s post-test).

(A) 293 or COS cells were incubated with luciferase-encoding Tpell phage at a MOI of 1×10⁶. Bafilomycin A was added to the culture medium at the indicated doses. 24 hours later the cells were washed, and placed in medium lacking the drug. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to bafilomycin A had no effect on phage-mediated luciferase gene expression. (B) 293 cells were pulsed with medium containing fluorescein (F) and tetramethylrhodamine (T) dextran in the presence or absence of bafilomycin A for 1.5 hrs. Cells were washed and fresh media was added with or without drug. 2 hours later, cells were washed, trypsinized and resuspended in fresh media for flow cytometric analysis of F and T fluorescence; the ratio of T (pH sensitive) to F (pH insensitive) fluorescence was then calculated (van Weert et al., 1995).

293 cells were incubated with luciferase-encoding WT or Tpell phage at a MOI of 1×10⁶. The indicated drugs were added to the culture medium at doses of 10 μM (CA-074, cathepsin B inhibitor; CatB), 10 μM (cathepsin L inhibitor III; CatL), 10 μM and 10 μM (CA-074, cathepsin B inhibitor plus cathepsin L inhibitor III, respectively; CatB+L). 16 hours later the cells were washed, and placed in medium lacking the drugs. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to CatB or CatL inhibitors alone resulted in a modest increase in phage-mediated luciferase gene expression (that did not acheive statistical significance). In contrast, treatment of cells with a combination of the CatB and the CatL inhibitor resulted in a statistically significant enhancement of phage-mediated gene transfer (the asterisk denotes p < 0.05, when compared to untreated cells, as determined by one-way ANOVA with Tukey’s post-test).

Luciferase-encoding lambda phage particles were generated, displaying either a wild-type major coat protein, gpD (WT) or a modified form of gpD ("Tpell"). Phage particles were then added to COS cells at a MOI of 1×10⁶, and cells were incubated in the presence of the proteasome inhibitors, lactacystin (3 μM), bortezomib (10 nM) or MG132 (1 μM) as described in the legend to Fig. 1. 16 hours later, the cells were washed, and the culture medium was replaced with medium that did not contain proteasome inhibitors. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to the various proteasome inhibitors enhanced phage-mediated luciferase gene expression. This result acheived statistical significance for the Tpell phage, indicated by the asterisk, in the case of bortezomib (one way ANOVA; p value <0.05, when comparing bortezomib treated cells to the untreated control cells); a strong trend is also apparent for MG132 and lactacystin.

Luciferase-encoding lambda phage particles were generated, displaying either a wild-type major coat protein, gpD (WT) and a recombinant form of gpD, bearing a PEST motif (SPAETPESPPATPK; phage particles displaying this peptide are hereafter designated “Tpell”). Phage particles were then added to 293 cells at a multiplicity of infection (MOI) of 1×10⁶, and cells were incubated in the presence of the proteasome inhibitors, lactacystin (3 μM), bortezomib (10 nM) or MG132 (1 μM). 16 hours later, the cells were washed, and the culture medium was replaced with medium that did not contain proteasome inhibitors. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to the various proteasome inhibitors resulted in an increase in phage-mediated luciferase gene expression. This result acheived statistical significance for both WT and Tpell phage, indicated by the asterisk, in the case of MG132 (one way ANOVA; p value <0.05, when comparing MG132 treated cells to the untreated control cells); a strong trend is also apparent for lactacystin and bortezomib.

Phage particles was exposed to 0, 50 or 100 ng of purified cathepsin L (catL). After inactivation of catL, the infectivity of the wild type and Tpell phage particles was determined by titering on E. coli host cells (A). In addition, the integrity of the phage particles was also assessed, by adding DNase to the catL-exposed phage particles. After DNase treatment, the phage particles were lysed and their DNA content was collected, and subjected to agarose gel electrophoresis. Shown is a photograph of an ethidium bromide (EtBr)-stained gel that was loaded with DNA extracted from equivalent amounts of the indicated phage, following exposure to DNase (B). The results show that incubation with catL resulted in a dose-dependent decline in the infectious titer of the phage (A), and damage to the capsid, resulting in exposure of the phage genomic DNA - and rendering it susceptible to degradation by DNase (B). In panel B, NT denotes “no treatment with catL” and MWM denotes “molecular weight markers”. The high molecular weight DNA stained by EtBr represents intact, linear λ phage genomic DNA (approx. 50 kb in length).

293 cells were incubated with luciferase-encoding Tpell phage at a MOI of 1×10⁶, in the presence or absence of bortezomib (10 nM). 16 hours later the cells were washed, and placed in medium lacking the drug. 24 hours following addition of phage, the cells were harvested, fractionated and lysed. Nuclear phage DNA levels were then quantitated by DNA PCR analysis using a TaqMan® primer/probe set specific for the lambda phage integrase gene. Phage DNA levels were normalized to measured levels of cellular DNA (18S rRNA DNA), and are reported here as copies of lambda phage genomic DNA per 293 cell. The analysis was performed in triplicate (three separate wells of cells), and results are presented as the mean of these results; the bars represent the standard error of the mean. Treatment of cells with bortezomib resulted in a statistically significant increase in nuclear accumulation of phage DNA (p < 0.01, when compared to untreated cells; one-way ANOVA with Tukey’s post-test).

GFP-encoding Tpell phage were added to 293 cells at a MOI of 1×10⁶. The indicated drugs were added to the culture medium at doses of 10 nM (bortezomib; bort.) or 1 μM (MG132). 16 hours later the cells were washed, and placed in medium lacking the drugs. 48 hours following addition of phage, the cells were harvested and GFP expression was measured by flow cytometric analysis. The analysis was performed in triplicate (three separate wells of cells), and results are presented as the mean percentage of GFP positive cells (A) and the mean fluorescence intensity (MFI) of GFP expression (B). The bars represent the standard error of the mean. Treatment of cells with MG132 resulted in a statistically significant increase in both the percentage of GFP positive cells (**; p < 0.01, when compared to untreated cells; one-way ANOVA with Tukey’s post-test) and the MFI of GFP expression (*; p < 0.05, when compared to untreated cells; one-way ANOVA with Tukey’s post-test). Treatment of cells with bortezomib also resulted in a statistically significant increase in the MFI of GFP expression (**; p < 0.01, when compared to untreated cells; one-way ANOVA with Tukey’s post-test) and a more modest increase in the percentage of GFP positive cells (which did not achieve statistical significance).

293 cells were incubated with luciferase-encoding Tpell phage at a MOI of 1×10⁶. The indicated drugs were added to the culture medium at doses of 10 μM (CA-074, cathepsin B inhibitor; CatB), 10 μM (cathepsin L inhibitor III; CatL), 10 μM and 10 μM (CA-074, cathepsin B inhibitor plus, cathepsin L inhibitor III, respectively; CatB+L), 10 nM (bortezomib; bort.), 50 μM (chloroquine; CHQ); in some cases (as indicated), cells were also exposed to combinations of these agents (e.g., CaB/L+Bort., CHQ+Bort.). 16 hours later the cells were washed, and placed in medium lacking the drugs. 48 hours following addition of phage, the cells were harvested and lysed, and luciferase activity was measured. Exposure of cells to bortezomib alone (Bort.) or to CatB plus CatL inhibitors (CatB/L) alone resulted in a modest increase in phage-mediated luciferase gene expression, while treatment of cells with a combination of these agents (CatB/L+Bort.) resulted in a synergistic and statistically significant enhancement of phage-mediated gene transfer (the asterisk denotes p < 0.001, when compared to untreated cells or cells exposed to either agent alone, as determined by one-way ANOVA with Tukey’s post-test). Exposure of cells to a high concentration of chloroquine (CHQ) also resulted in a strong and statistically significant increase in phage-mediated gene transfer (p < 0.001, when compared to untreated cells; one-way ANOVA with Tukey’s post-test). However, cotreatment of cells with CHQ in combination with CatB plus CatL inhibitors did not result in any further increase in luciferase expression, compared to cells exposed to CHQ alone.