The primary antibodies used were: p-Stat3 (9145) from Cell Signaling Technology, Inc. (Beverly, MA, USA); glutathione S-transferase (GST; sc-33614) and Rac1 (sc-217) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); FLAG (T510-2) and green fluorescent protein (GFP; T508-2) from Signalway Antibody (Baltimore, MD, USA); Myc-Tag (AM1007a) from Abgent (San Diego, CA, USA); anti-bromodeoxyuridine (BrdU; 560810) from BD Biosciences (San Jose, CA, USA) and Stat3 (51076-2-AP) from Proteintech Group (Chicago, IL, USA). Other antibodies used, including FITC-conjugated goat anti-mouse, Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 594-conjugated goat anti-mouse antibodies and HRP-conjugated goat antibodies against rabbit and mouse, were purchased from Jiayuan Biotech Co. Ltd. (Wuhan, China).

The point mutation for tryptophan and the mutation of WVLGE to AVLGA were generated by overlapping PCR using primers as follows:i) point mutation for tryptophan of SGLP, N-terminus 5′-ATGGATCCAAATCATTTGCAGAGCCAATTC-3′ and 5′-GTTCACCCAAGACCCATTTAATGGCATC-3′ and C-terminus 5′-GCCATTAAATGGGTCTTGGGTGAA CAAT-3′ and 5′-CACCGAATTCCTCAATTTCAAATT CTTTTAGTTTG-3′ and ii) point mutation for WxxxE to AxxxA, N-terminus 5′-GATATCAAATCATTTGCAGAGCCAAT-3′ and 5′-TGTGCACCCAAGACCGCTTTAATG-3′ and C-terminus 5′-AAAGCGGTCTTGGGTGCACAATC-3′ and 5′-GCGAATTCCTACTCAATTTCAAATTC-3′. SGLP sequences were cloned into pGEX4T1, pEGFP-C1 and pCDF1-MCS2-EF1-Puro vectors (System Biosciences, Mountain View, CA, USA). In addition, the FLAG-tag DYKDDDDK and Kozak sequences were inserted into the pCDF1-MCS2-EF1-Puro vector. DN-Rac1 (T17N) and CA-Rac1 (Q61L) pRK5 plasmids were purchased from Addgene Inc. (Cambridge, MA, USA).

HEK 293T and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) and supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37°C in a humidified 5% CO₂ atmosphere. Lentiviral packaging was conducted in HEK 293T cells using pPACK™ Packaging mix (System Biosciences) according to the manufacturer’s instructions. The lentiviral titers were determined by an UltraRapid Titering kit (System Biosciences). HeLa cells were transduced with SGLP lentiviral particles with 6 μg/ml polybrene (Santa Cruz Biotechnology, Inc.) for 12 h. Transduced cells were selected with 2 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA) for two weeks and the stable cell lines were maintained in growth medium with 1 μg/ml puromycin. Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) was used for transfection of plasmids and small interfering RNAs according to the manufacturer’s instructions.

GST-tagged protein expression was induced by adding 0.1 mM isopropyl-β-d-thiogalactopyranoside to BL21 E. coli cultures at 30°C for 6 h. GST-tagged proteins were purified by affinity chromatography with glutathione Sepharose™ 4B beads (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s instructions. HeLa cells (~1×10⁷) were treated with 100 ng/ml IL-6 for 24 h prior to harvesting. Equal quantities of precleared lysates (~2 mg) were incubated with sepharose beads and 10 μg GST or GST-SGLP for 2 h at 4°C. The beads were washed three times with washing buffer (pH 7.2, 10 mM NaPO₄, 10 mM NaN₃, 150 mM NaCl and 0.1% Tween-20). The bait and pray proteins were then eluted from beads with elution buffer (pH 8.0, 50 mM Tris-Cl and 30 mM GSH) and subjected to western blot analysis. The active Rac1 pull-down assay was conducted according to a modified method from Teng et al(13). The GST-Pak1-PBD, which contains the peptide ISLPSDFEHTIHVGF (CRIB domain), was purchased from Millipore (Billerica, MA, USA) and active Rac1, as well as total Rac1, were probed separately. For immunoprecipitation, the GFP- or GFP-SGLP-transfected HeLa cell lysates (1 mg) were precleared with 25 μl protein G sepharose beads (Amersham Biosciences) and incubated with 2 μg primary antibodies at 4°C for 2 h, followed by incubation with 25 μl protein G sepharose beads at 4°C for 2 h. Western blot analyses were performed as previously described (21).

Immunofluorescence images were captured using an Olympus FluoView™ FV1000 confocal microscope. HeLa cells expressing SGLP and vector control were labeled with 250 nM Mitotracker at 37°C for 15 min. The cells were then fixed, permeabilized and stained with 50 μg/ml fluorescein isothiocyanate (FITC)-phalloidin at room temperature for 2 h followed by DAPI counterstaining. The ImageJ plugins for colocalization analysis are described and may be downloaded at http://fiji.sc/wiki/index.php/Colocalization_Analysis. The colocalization of FLAG-SGLP with Stat3 was assessed with the ImageJ plugin as described by Fay et al(22) and the Pearson’s correlation coefficients were also calculated using ImageJ. For FRET analysis, images were recorded in three channels for donor, acceptor and transfer, respectively. The donor and acceptor bleed-through was determined and the pseudocolor image of FRET was captured using the ImageJ plugin ‘FRET and Colocalization Analyzer’, as described by Hachet-Hass et al(23). Photobleaching of DsRed-Rac1 was achieved by continuous excitation at 561 nm with full-lamp intensity. Images of GFP-SGLP and DsRed-Rac1 were recorded at intervals of 30 sec.

Transwell assays were conducted using Transwell migration chambers with an 8-μm pore size (Corning Inc., Acton, MA, USA) according to the manufacturer’s instructions. HeLa cells (~1×10⁵) suspended in 500 μl of serum-free DMEM were seeded in each chamber. The migration chamber was placed into a 24-well plate with 500 μl DMEM containing 10% FBS and incubated at 37°C with 5% CO₂. Following 8 h, cells on the upper surface were removed with a cotton swab. The migrated cells on the lower surface were fixed and stained with 1% crystal violet. Five microscopic fields (magnification, ×200) were counted per filter in three independent experiments.

HeLa cells were incubated with 50 μM BrdU at 37°C for 30 min followed by fixation in 80% ethanol at 4°C overnight. The cells were sequentially incubated with 2 M HCl (room temperature, 30 min) and 0.1 M Na₂B₄O₇ buffer (pH 8.5) for DNA denaturation. Following extensive washing, the cells were immunostained at room temperature with an anti-BrdU antibody (1:50) for 2 h and a secondary FITC-conjugated antibody (1:200) for 1 h. The cells were then resuspended in phosphate-buffered saline containing 50 μg/ml propidium iodide and 125 μg/ml RNAase, prior to flow cytometry.

Analysis of data from three independent experiments were conducted using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) and expressed as the mean ± SD. Student’s t-test was used for comparisons among groups. P<0.05 was considered to indicate a statistically significant difference.

As SGLP possesses a WxxxE motif, which is required for a number of bacterial virulence factors in subverting host cellular actin cytoskeleton, the actin filament patterns in HeLa cells expressing SGLP or vector control by FITC-phalloidin staining were examined. The actin cytoskeleton was observed to be markedly altered following transduction of HeLa cells with lentiviral particles expressing SGLP, which was characterized by reduced stress fibers and increased lamellipodia and filopodia (Fig. 1). As the Rho family members of small GTPases are key regulators of stress fibers, lamellipodia and filopodia (24), this result indicated that SGLP is associated with functions of host Rho small GTPases.

The results presented in Fig. 1 revealed that SGLP, which resembles a number of bacterial virulence factors that contain a WxxxE motif, was associated with Rho-family small GTPases. One of the members of Rho small GTPases is Rac1, which is a binding partner of Stat3 (8). In addition, using computational analysis, SGLP was observed to possess a coiled-coil domain that may interact with the corresponding coiled-coil domain of Stat3 (20). Therefore, it was hypothesized that there are probable functional and/or physical interactions of SGLP with Rac1 and Stat3. Thus, a possible interaction between SGLP and Rac1 and Stat3 was investigated.

A GST pull-down assay revealed that the purified GST-SGLP protein binds to host endogenous Rac1 and Stat3 (Fig. 2A). In addition, in HeLa cells, endogenous Rac1 and Stat3 were co-immunoprecipitated with GFP-SGLP using a GFP antibody (Fig. 2B). Immunofluorescence analyses revealed that FLAG-tagged SGLP colocalized with Stat3 in the nucleus and cytoplasm (Fig. 2C). The Pearson’s correlation coefficient of colocalization between SGLP and Stat3 was ~0.75 as measured by ImageJ, whereas that of the control was <0.2 in 9 cells from three independent experiments. GFP-SGLP also colocalized with DsRed-Rac1 in HeLa cells coexpressing these two proteins (Fig. 2D). Although the DsRed-Rac1 distribution was homogeneous throughout the cell, which makes it difficult to interpret for colocalization directly, the FRET between GFP-SGLP and DsRed-Rac1 revealed a significant colocalization of SGLP and Rac1 compared with the control (Fig. 2D). Furthermore, consecutive confocal images showed that the GFP-SGLP (donor) intensity increased following photobleaching of the DsRed-Rac1 (acceptor) (Fig. 2E). A linear correlation was observed between the intensity of donor recovery vs. acceptor photobleaching, indicating that GFP-SGLP interacts with DsRed-Rac1 (Fig. 2F).

The results in Fig. 2 revealed that SGLP interacts with Rac1 and Stat3, therefore the study focused on the effect of SGLP on the function of Rac1 and Stat3. As the WxxxE motif is required for a number of bacterial virulence factors to activate cellular Rho-family small GTPases, including Rac1 (16), it was hypothesized that SGLP may also activate Rac1 and that the WxxxE motif is crucial for this effect. Therefore the study investigated whether SGLP activates Rac1, as well as whether the WxxxE motif is responsible for the activation of Rac1. The wild-type (wt) WVLGE sequence within wt SGLP was mutated to AVLGA to generate an SGLP mutant. HeLa cell lines stably expressing wt SGLP, SGLP mutant or vector, as well as parental HeLa cells were studied. Active Rac1 levels were determined by a GST-Pak1 pull-down assay followed by western blot analysis. In the presence of the WxxxE motif, wt SGLP induced greater Rac1 activation in HeLa cells as compared with the SGLP mutant and the vector control, as well as the parental cells (Fig. 3A and C). In addition, western blot analysis revealed that wt SGLP increased the phosphorylation of Stat3 at tyrosine-705 residue (p-Stat3) in HeLa cells and that the SGLP mutant did not exhibit such a prominent effect on Stat3 phosphorylation (Fig. 3B and D).

Fig. 3 shows that wt SGLP increased cellular p-Stat3 levels, which reflects its role in the activation of Stat3. Thus, the mechanism by which SGLP activates Stat3 was investigated. As Rac1 mediates Stat3 activity through direct binding to Stat3 and an indirect activation loop of autocrine IL-6 (8,25), it was examined whether the SGLP-induced activation of Stat3 was dependent on Rac1 activity. The wt and mutant SGLP-transduced HeLa cells were transfected with dominant negative Rac1 (DN-Rac1) or constitutive active Rac1 (CA-Rac1) plasmids separately. Overexpression of DN-Rac1 was observed to eliminate the increase in p-Stat3 levels in wt SGLP-transduced HeLa cells (Fig. 4A), whereas overexpression of CA-Rac1 rescued the decrease in p-Stat3 levels in mutant SGLP-transduced HeLa cells (Fig. 4B). The results suggested that SGLP activates Stat3 depending on the active form of Rac1.

The aforementioned observations suggest that SGLP activates Rac1 and Stat3. Since Rac1 and Stat3 are involved in cell migration and proliferation (14), the possibility of whether SGLP may affect migration and/or proliferation of HeLa cells was determined. In a Transwell assay, wt SGLP was observed to increase the transwell migration of HeLa cells relative to the control and SGLP (mutant) (Fig. 5A), which is consistent with the levels of active Rac1 demonstrated in Fig. 3A. These observations suggest that the effect of wt SGLP on HeLa cell migration is associated with the wt SGLP-induced Rac1 activation. BrdU incorporation rates in HeLa cells were then investigated by flow cytometry. Wt SGLP was observed to promote BrdU incorporation (Fig. 5B), which suggests that SGLP may exert a pro-proliferative effect in HeLa cells.