Visualization of clustered data.

<p>(A) Visualization of a between-mouse factor. Each point represents the mean soma size of a mouse ± standard error (SE). [SE = standard deviation (mean soma size)]. (B) Visualization of a within-mouse factor. Each point represents the soma size of an individual neuron within a mouse. The colors correspond to the mouse to which the neurons belonged. Each mouse has neurons with control and Pten shRNA. (C) Visualization of an interaction effect between the within-mouse and between-mouse factor. The red dotted line represents the vehicle control mice and the blue solid line represents the fatty acid delivery mice. Pten knockdown status 0 = control shRNA and 1 = Pten shRNA. The black dotted line depicts the expected result if there were no interaction effect, and the space between the black dotted line and the blue line, denoted by the curly bracket, represents the size of the interaction effect.</p

Main decision points for statistical analysis of clustered data.

<p>The flow chart outlines the primary questions researchers should address when weighing options of statistical research design of a study with clustered data. *Readers should refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146721#pone.0146721.t002" target="_blank">Table 2</a> for subtler differences between the marginal and mixed-effect model.</p

Targeting Wall Teichoic Acid <i>in Situ</i> with Branched Polyethylenimine Potentiates β‑Lactam Efficacy against MRSA

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a medical concern. Here, we show that branched polyethylenimine (BPEI), a nontoxic, cationic polymer, restores MRSA’s susceptibility to β-lactam antibiotics. Checkerboard assays with MRSA demonstrated synergy between BPEI and β-lactam antibiotics. A time-killing curve showed BPEI to be bactericidal in combination with oxacillin. BPEI did not potentiate efficacy with vancomycin, chloramphenicol, or linezolid. When exposed to BPEI, MRSA increased in size and had difficulty forming septa. BPEI electrostatically binds to wall teichoic acid (WTA), a cell wall anionic polymer of Gram-positive bacteria that is important for localization of certain cell wall proteins. Lack of potentiation in a WTA knockout mutant supports the WTA-based mechanism. These data suggest that BPEI may prevent proper localization of cell wall machinery by binding to WTA; leading to cell death when administered in combination with β-lactam antibiotics. Negligible <i>in vitro</i> toxicity suggests the combination could be a viable treatment option

<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 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