<i>H. pylori</i> infection results in increased RKIP transcription and nuclear localization.

<p>(A) RKIP transcription reporter assay of AGS cells transiently transfected with RKIP luciferase construct and HA-RKIP for 24 h, then co-cultured with <i>H. pylori</i> for 12 h in the presence or absence of the PKC inhibitor. In comparison to empty vector controls, relative transcriptional activity was significantly increased for * <i>H. pylori</i>, p<0.002; and ** <i>H. pylori</i>+RKIP, P<0.0003. Comparing the loss of relative luciferase activity of <i>H. pylori</i> and RKIP when compared to <i>H. pylori</i>, RKIP and Bis, p<0.0004. Data represents the mean +/− standard deviation (sd) of the fold increase relative to empty vector controls in 2 independent experiments performed in duplicate. (B) Western blot analysis of nuclear and cytosolic fractions of AGS cells co-cultured with <i>H. pylori</i> for 4 h for the expression of pRKIP and total RKIP. Actin and laminA provide verification of successful cytoplasmic and nuclear fraction separation.</p

The pathogenicity island of <i>H. pylori</i> is responsible for RKIP phosphorylation.

<p>Western blot analysis of (A) AGS cells co-cultured with <i>H. pylori</i> strains for 6 h and examined for the indicated proteins. C = control (uninfected), WT = AGS cells infected with wild type <i>H. pylori</i> for 6 h, <i>PAI</i>- and <i>oipA</i>- represent isogenic mutants lacking these genes. (B) AGS cells transiently transfected for 24 h with RKIP S153 cDNA or 24 h and co-cultured with <i>H. pylori</i> for 6 h. (C) RKIP luciferase reporter assay of AGS cells transiently transfected with S153V RKIP in the presence or absence of <i>H. pylori</i> infection. In comparison to empty vector controls, the relative activity of RKIP transcription was increased by: *<i>H. pylori</i>, p<0.002; **RKIP, p<0.002; ***S153V, p<0.03, ****<i>H. pylori</i> and RKIP, p<0.0005; *****<i>H. pylori</i> and S153V, p<0.003. **, RKIP transcriptional activity was significantly decreased by the S153V compared with the wild type RKIP construct in response to <i>H. pylori, #</i>, p<0.0003. The data represents the mean +/− sd of 2 independent experiments performed in duplicate.</p

<i>H. pylori</i> targets RKIP for proteasomal degradation and results in the induction of Snail.

<p>AGS cells were (A) treated with MG132 and examined for the indicated proteins via Western blot analysis. V represents vehicle (DMSO) control. (B, C) Cells were co-cultured with <i>H. pylori</i> for the indicated times and examined for the expression of Snail or (D) measured for Snail mRNA by real-time PCR at the indicated times after <i>H. pylori</i> infection.</p

RKIP enhances <i>H. pylori</i> mediated apoptosis.

<p>(A) Western blot analysis of AGS transiently transfected with RKIP for 24 h then infected with <i>H. pylori</i> for 6 h. Densitometry analysis for the average of 2 independent experiments indicated a 1.53 fold increase in apoptosis (average intensity of 0.19 vs 0.295) in <i>H. pylori</i> infected cells; 2.1 fold increase (average intensity of 0.19 vs 0.403) in cells transfected with RKIP; a 2.6 fold increase (average intensity of 0.19 vs 0.495) in cells infected with <i>H. pylori</i> and transiently transfected with RKIP when normalized to actin. In parallel apoptosis was measured by (B) flow cytometry. C) An ELISA based DNA fragmentation assay was used to measure apoptosis. Compared to empty vector control in (B) apoptosis was increased by <i>* H. pylori</i>, p<0.0008; ** RKIP, p<0.003; *** <i>H. pylori</i> and RKIP, p<0.0005. In (C) compared to empty vector controls, apoptosis was significantly increased by: * <i>H. pylori</i>, p<0.000063; ** RKIP, p<0.006; <i>*** H. pylori</i> and RKIP, p<0.0007. The data for B and C represents the mean +/− sd of 2 independent experiments performed in duplicate. (D) Western blot analysis of AGS cells infected with Lentivirus to knock down RKIP expression prior to 6 h infection with <i>H. pylori</i>. CTR =  uninfected AGS cells, HP =  AGS cells infected with <i>H. pylori</i>, KD =  AGS cells infected with lentivirus to knockdown RKIP, KD+RKIP AGS cells infected with lentivirus to knockdown RKIP and infected with <i>H. pylori</i> for 16 h. (E) Apoptosis (by DNA fragmentation ELISA) was measured after 16 h of <i>H. pylori</i> infection. Apoptosis was significantly increased by <i>H. pylori</i> in AGS cells *p<0.0007; and in AGS cells with RKIP knockdown, **p<0.003 and was decreased comparing <i>H. pylori</i>-infected RKIP knockdown AGS cells with <i>H. pylori</i>-infected parental AGS cells, #p<0.0006. The data shown represents the mean +/− sd of 2 experiments performed in triplicate.</p

IL-6 promotes RKIP and STAT 3 transcription and phosphorylation; <i>H. pylori</i> infection induces STAT3 transcription and phosphorylation.

<p>(A) Western blot analysis of AGS cells treated with the indicated concentrations of IL-6 and for 6 hours. Densitometry was performed and for pRKIP expression, our results indicate a 1.8 fold incerase of pRKIP (average intensity 0.59 vs 1.051) in cells treated with 25 ng/ml IL-6 and a 1.35 fold increase (average intensity 0.59 vs 0.772) in cells treated with 50 ng/ml IL-6. For RKIP expression, our results indicate a 0.8 fold incerase of pRKIP (average intensity 0.69 vs 0.563) in cells treated with 25 ng/ml IL-6 and a 1.05 fold increase (average intensity 0.69 vs 0.745) in cells treated with 50 ng/ml IL-6 when normalized to actin at each time point. (B) Western blot analysis of AGS co-cultured with <i>H. pylori</i> and examined for pY705 STAT3 for the indicated times; (C) STAT3 luciferase reporter transcriptional assay of AGS cells co-cultured with <i>H. pylori</i> at the indicated MOI; (D) STAT3 luciferase reporter assay of AGS cells transiently transfected with STAT3 for 24 h and co-cultured with <i>H. pylori</i> and/or treated with IL-6 for 6 h. A paired t-test was performed to analyze the increase or decrease in STAT3 transcription of experimental samples when compared to empty vector (EV): *IL-6, p<0.0003; **STAT3, p<0.009; *** <i>H. pylori</i> p<0.0005; **** STAT3 and <i>H. pylori</i> p<0.0000023.</p

<i>H. pylori</i> infection results in RKIP phosphorylation.

<p>(A) Western blot analysis of AGS cells co-cultured with <i>H. pylori</i> (HP) at MOI of 100∶1 for 2 and 6 h and examined for pRKIP, RKIP, and actin expression. Densitometry was performed on three independent experiments and band intensities normalized in comparison to Actin for each time point. Our results indicated a 3.126 fold increase (average intensity 0.44 vs 1.376) of pRKIP after 2 h and 1.384 fold increase (average intensity 0.6774 vs 0.938) of RKIP after 2 h of <i>H. pylori</i> infection. (B) AGS cells co-cultured for 6 h in the presence or absence of the PKC inhibitor bisindolylmaleimide (Bis), were examined for the expression of pRKIP, RKIP and actin.All treatments were performed in 1% DMSO as a vehicle control (Bis).</p

RKIP is required to inhibit STAT3 activation and breast tumor xenograft growth.

<p>(A) STAT3 luciferase reporter activity was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092478#pone-0092478-g002" target="_blank">Figure 2</a> in cells treated with 40 ng/ml IL-6 for 6 h. <i>A</i>, represents MDA RKIP cells and (B) MDA si175 RKIP knockdown cells. Paired t-test was performed; *(p<0.007) in IL-6 treated cells when compared to EV control. Luciferase reporter activities for duplicate of 2 representative experiments +/- s.d. are shown. (C) Western blot analysis was performed in MDA RKIP and MDA si175 RKIP treated with 40 ng/ml IL-6 for 2 h. Whole cell lysates were examined for the expression of the indicated proteins. (D) MDA cells were implanted s.c. in the dorsal flanks of Nu/J female mice. Tumors were measured using calipers twice weekly, and volumes were calculated using the formula: weight (mg)  =  [width (mm)² × length (mm)]/2. A paired t-test was performed indicating the decrease of RKIP *(p<0.002) or increase of **(p<0.008) si175RKIP, ***(p<0.002) STAT3 of tumor growth when compared to EV. A decrease in tumor growth was detected between STAT3 and RKIP/STAT3 ****(p<0.009). Mean tumor size for each treatment group is plotted (<i>n</i>  =  6) +/− s.d.</p

RKIP inhibits Src-STAT3 interaction.

<p>(A), top panel, Cartoon depicting a series of RKIP deletion constructs that were examined for their ability to inhibit c-Src- and JAK1-mediated STAT3 activation. <i>Middle panel</i>, Some of the deletion constructs were transfected in the presence of c-Src or JAK1 into cells for 48 h. Whole cell protein lysates were examined for the expression of STAT3, STAT3 pY⁷⁰⁵ and actin. <i>Lower panel</i>, Cells were transfected with EV, c-Src or RKIP deletion constructs N93 and C93. After 48 h, cells were harvested and whole cell lysates prepared to examine the expression of the indicated proteins via Western blot analysis. (B) Cells were transfected with c-myc EV, or the following tagged expression plasmids: myc-c-Src (2 μg), HA-STAT3 (2 μg), Flag-RKIP (4 μg) or the combination for 48 h. Cells were lysed in RIPA buffer, cellular debris removed, a portion of the sample removed as the IP input and the remaining supernatant incubated and rotated with an antibody to c-myc for 4 h at 4°C. Subsequently, protein G agarose was added and the samples rotated overnight at 4°C. Protein G agarose containing immuno complexes was extensively washed, 2× sample buffer added and the samples heated at 95°C for 5 min. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose. The samples subjected to IP were divided in half. One filter was examined with an antibody to HA the other with an antibody to c-myc. The HA filter was stripped and examined for STAT3 levels. The input samples were examined for the expression of the indicated proteins. (C) Western blot analysis of cells transfected with EV (lane 1), c-Src (lane 2), HA-RKIP (lane 3) and c-Src + HA-RKIP (lane 4) to examine the expression of the indicated proteins that were separated on 15% SDS-PAGE. (D) RKIP inhibits c-Src-mediated MDA-231 cell migration. MDA-231 cells were transfected with empty vector, c-Src, c-, HA-RKIP, RKIP siRNA, scrambled siRNA or the combination. The cells were allowed to grow to near confluence. Cells were wounded with a pipette and after 24 h the migrated cells were fixed then stained with crystal violet and quantified. A paired t-test was performed indicating *(p<0.0002) an increase in migration in c-Src transfected MDA cells when compared to EV transfected cells and **(P<0.005) inhibition of c-Src-mediated increase in migration when compared to cells transfected with c-Src and RKIP. The data represents two independent experiments performed in triplicate.</p

Microtubule inhibitors cause cell cycle arrest and induce RKIP expression.

<p>(A) DU145 and PC3 cells were treated with ENMD-1198 or MKC-1. Cells were harvested and washed twice with PBS and whole cell lysates were prepared for Western blot analysis as we have previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092478#pone.0092478-AlMulla1" target="_blank">[28]</a>. Proteins (unless indicated 50 μg/sample was used for Western blot analysis) were separated by 10% SDS-PAGE, transferred to nitrocellulose and analyzed with antibodies to the indicated proteins. <i>Note: for all Western blots described in this Figure legend and for all other subsequent Figure legends, the exposure time used to identify the various proteins was variable</i>. (B) DU145 cells were treated with 200 nM ENMD-1198 or MKC-1 for 6 or 12 h. Cells were prepared for Western blot analysis for the indicated proteins. (C) DU145, PC3 prostate and MDA breast cancer cells (1 × 10⁵) were used for FACS analysis after treatment with the indicated concentrations of ENMD-1198 or MKC-1. Cells were harvested, washed twice with phosphate buffered saline (PBS) and prepared for flow cytometry as previously described (43). Analysis for apoptosis was performed using a FACSCalibur flow cytometer (Data represents the mean ± S.D., of 2 independent experiments performed in duplicate). (D) (Left panel) PC3 and MDA cells were transiently transfected with HA-RKIP expression plasmid for 48 h. Whole cell lysates were prepared and Western blot analysis was performed as described in A for the indicated proteins. <i>Note for this and all other subsequent experiments described where RKIP was transfected into cells, the lower RKIP band represents endogenous RKIP, while the upper RKIP band represents ectopic RKIP (HA-RKIP)</i>. (Right panel), DU145 cells were treated with ENMD-1198 or MKC-1 for 24 h. Whole cell lysates were prepared and Western blot analysis was performed for the indicated proteins. (E, top) DU145 cells were transiently transfected with various combinations with expression plasmids for RKIP, RKIP siRNA, scramble siRNA or empty vector (EV) for 48 h. The cells were treated with 200 nM ENMD-1198 for 24 h. Cells were harvested and analyzed for apoptosis as described in (C). A paired t-test was performed to analyze the increase in apoptosis in cells when compared to empty vector (EV): *(p<0.001) transiently transfected with RKIP; **(p<0.00007) treated with ENMD-1198; ***(p<0.0002) with the combination of RKIP transfection and ENMD-1198 treatment. Scrmbl = scrambled siRNA. A decrease in apoptosis was observed ****(p<0.000012) in cells treated with ENMD-1198 when compared to cells transiently transfected with RKIP siRNA and treated with ENMD-1198. (Data represents the mean ± S.D., of 2 independent experiments performed in triplicate). (E, bottom) Western blot analysis of the same experimental conditions described in Fig. 2E, top. The Western blot analysis was repeated 3 times and the Figure is representative of one of the experiments. Densitometry was performed and the relative intensity (fold increase) when compared to Actin for lanes 1-8, respectively were 0.78, 1.7, 0.19, 0.84, 0.99, 0.67, 0.36 and 0.92.</p

RKIP blocks JAK and Raf-mediated STAT3 activation.

<p>(A) RKIP inhibits JAK1, 2-triggered STAT3 activation. 293T cells were transfected with the Luc reporter construct containing 2× STAT3 binding SIE-fragment and co-transfected with either JAK1, 2, 3 or TYK2 in the presence or absence HA-RKIP and ENMD-1198. Cells were placed in serum free medium and cell extracts were subjected to luciferase assay to estimate transcriptional activity. Averages and standard deviations (error bars) of the values of luciferase activities from triplicates of 3 representative experiments are shown. (B) Western blot analysis demonstrating inhibition of STAT3 pY⁷⁰⁵ phosphorylation in HA-RKIP and JAK1 or 2 transiently transfected 293T cells. Raf-mediated STAT3 pY⁷⁰⁵ phosphorylation is inhibited by RKIP: (C) 293T cells were transfected with Raf-BXB, CMV empty vector or HA-RKIP. After 48 h cells were left untreated or exposed to IL-6 for 1 h. Cell lysates were analyzed by Western blot. (D) 293T cells were transfected with the STAT3 luciferase reporter construct and/or with EV, Raf-BXB or HA-RKIP. After 24 h, some cells were treated with PD98059 (PD) and after 48 h from the initial time of transfection, some cells were treated with IL-6 for 6 h. A paired t-test was performed indicating *(p<0.000005) increase in BXB transfected cells when compared to EV; decrease in STAT3 transcriptional activity **(p<0.00006) in PD treated cells transfected with BXB and ***(p<0.00004) in RKIP and BXB transfected cells when compared to the cells transfected with BXB. Luciferase reporter activities for triplicates of 3 representative experiments +/- s.d. are shown. Western blot analysis of cells transfected with the indicated proteins in the presence or absence of PD treatment.</p