Intracellular localization and stability of the T3SS injected TALENs.

<p>(<b>A</b>) HeLa-Venus cells were infected with the indicated strains at an MOI of 100 for 3 hours. Nuclear proteins were extracted and subjected to SDS PAGE and Western blot by anti-Flag antibody; (<b>B</b>) HeLa-Venus cells were infected with PAK-JΔ<i>STY</i>/pExoS54-F-TALEN1 and PAK-JΔ<i>STY</i>/pExoS54-F-TALEN2 at MOI of 100 each for 3 hours. At the indicated time after the termination of infection, cytoplasmic and nuclear protein extracts were prepared and subjected to SDS PAGE and Western blot assay using anti-Flag Ab. NI, no infection control.</p

Loss of fluorescence in HeLa-Venus cells following bacterial delivery of TALEN proteins.

<p>(<b>A</b>) FACS analysis of fluorescence cell population three days after the delivery of indicated TALENs by <i>P. aeruginosa</i>; (<b>B</b>) Sequence changes in the TALEN targeting region among Venus negative cells.</p

Improvement of TALEN targeting efficiency.

<p>(<b>A</b>) Multiple rounds of TALEN injection. HeLa-Venus cells were infected with PAK-JΔ<i>STY</i>/pExoS54-F-TALEN1 and PAK-JΔ<i>STY</i>/pExoS54-F-TALEN2 at MOI of 100 each for 3 hours. Bacteria were cleared and cells were cultured for 24 hours, then repeated injection at MOI of 50 or 100. The FACS data of infected cells were compared by paired t-test and ANOVA. *P<0.05; **P<0.001. Error bars indicate standard deviations of triplicate assays. (<b>B</b>) HeLa-Venus cells were either left unsynchronized (U) or synchronized (S) through double thymidine blocking and released to determine the duration of the cell cycle by FACS analysis. (<b>C</b>) HeLa-Venus cells were synchronized to S phase (S) or unsynchronized (U) and infected by PAK-JΔ<i>STY</i>/pExoS54-F-TALEN1 and PAK-JΔ<i>STY</i>/pExoS54-F-TALEN2 at MOI of 100 each for 3 hours. The FACS data of infected cell populations were compared by paired or two-sample t-test. *P<0.05; **P<0.001. Error bars indicated the standard deviations of triplicate assays.</p

Plasmid transfection mediated delivery of TALENs into HeLa-Venus cells.

<p>(<b>A</b>) Fluorescent intensity of HeLa-Venus cells transfected by the indicated TALEN encoding plasmid constructs. Cells were analyzed by flow cytometry 3 days after the transfection. (<b>B</b>) Sequence changes in TALEN target region among the Venus negative cells.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Bacterial secretion of TALEN fusion protein.

<p>(<b>A</b>) Diagram of TALEN binding sites on <i>venus</i> gene. (<b>B</b>) Diagram of ExoS54-Flag-TALEN fusion protein; NLS, nuclear localization sequence. (<b>C</b>) Secretion profiles of laboratory strains of PAK-JΔ<i>STY</i> containing pUCP19 vector or PAK-JΔ<i>pscF</i>, PAK-JΔ<i>popD</i> and PAK-JΔ<i>STY</i> harboring pExoS54-F-TALEN1. Strains were grown under type III secretion inducing condition and culture supernatants and pellets were subjected to SDS PAGE followed by Western blot using anti-Flag antibody.</p

Protein injection into mouse embryonic stem cells (mESC).

<p>(<b>A</b>) mESC were infected with PAK-J strains at a MOI of 50 for 2.5 hours, lysed and assayed for injected ExoS-Flag by anti-Flag Western Blot. (<b>B</b>) mESC were infected with PAK-JΔ<i>STY</i>(pExoS-Flag) for 2.5 hours and subsequently fixed and immunostained with anti-Flag to illuminate translocated ExoS-Flag protein. (<b>C</b>) R26R-EYFP mESC cells contain a floxed terminator preventing downstream EYFP expression. (<b>D</b>) R26R-EYFP were infected with PAK-JΔ<i>STY</i>(pExoS54-Cre) at various MOIs for 2.5 hours. Cells were collected for FACS analysis 48 hours post infection. (<b>E</b>) EYFP-positive mESC were plated after infection to assess EYFP expression by confocal fluorescence microscopy.</p

Injection of indicated ExoS-Cre fusions.

<p>(<b>A</b>) Cre recombinase was fused in frame with various N-terminal portions of ExoS, with a nuclear localization sequence (NLS) in the fusion junction. (<b>B</b>) Detection of the five ExS54-Cre fusion proteins in PAK- JΔ<i>STY</i> harboring the corresponding fusion constructs. Bacterial cells were grown under type III inducing condition and cell lysates were subjected to Western blot using anti-Cre antibody. (<b>C</b>) TE26 cells were infected with PAK-JΔ<i>STY</i> containing the aforementioned constructs, lysed, and subject to Western Blot with anti-Cre antibody. N, no infection control; V, vector control, PAK-JΔ<i>STY</i>/pUCP20; P, T3SS mutant control, PAK-JΔ<i>popD</i>/pExoS54-Cre; Lanes1-5 are Cre fusions to ExoS17, ExoS54, ExoS96, ExoS234 and ExoS full length, respectively.</p

Assessment of Cre recombinase activity.

<p>Cre function was assessed by LacZ positive TE26 cells which contain a floxed terminator preventing <i>lacZ</i> expression (<b>A</b>). TE26 cells were infected for various times and MOIs and subsequently stained with X-gal for β-galactosidase (<b>B</b>) to determine the optimal infection conditions as indicated by percentage LacZ positive cells (<b>C</b>). TE26 cells were infected at an MOI of 50 for 3 hours before or after cell cycle synchronization and X-gal stained for β-galactosidase activity (<b>D</b>).</p

PCR primers used in this study.

<p>PCR primers used in this study.</p