Journal: PLoS ONE

Article Title: Identification of Candidate Serum Biomarkers for Schistosoma mansoni Infected Mice Using Multiple Proteomic Platforms

doi: 10.1371/journal.pone.0154465

Figure Lengend Snippet: (A) Scatterplot showing the discrimination of the early infection and control groups using m/z 3720 at F6ISL ( F raction 6 , IMAC chip, L ow laser intensity). Blue dots: control. Red dotes: early stage of infection. (B) Scatterplot showing the discrimination of the acute and chronic groups using m/z 13407.2 at F6CSH ( F raction 6 , CM10 chip, H igh laser intensity). Blue dots: acute. Red dotes: chronic. (C) Example of a candidate biomarker increased in infected mice over time. Serum SELDI-TOF MS mass spectra obtained for F6ISL ( F raction 6 , IMAC chip, L ow laser intensity) from infected mice (top 3 spectra) versus non-infected mice (bottom spectrum). A candidate biomarker as 7081 Da is gradually up-regulated from 3, to 6, to 12 weeks after infection (P value 0.006). (D) Example of a candidate biomarker increased in infected mice regardless the parasite burden. Serum SELDI-TOF MS mass spectra obtained for F6CSL ( F raction 6 , CM10 chip, L ow laser intensity) from infected mice (bottom 4 spectra) versus non-infected mice (top spectrum). A candidate biomarker as 5566.31 Da is up regulated in infected mice dependent on the infection rather than the dose of infected agents (P value 0.006). G1 infected with 200 cercariae, G2 infected with 150 cercariae, G3 infected with 100 cercariae, G5 infected with 50 cercariae.

Article Snippet: Biomarker Patterns SoftwareTM algorithms (Bio-Rad) were then used to analyze the peak intensities and to generate classification ‘trees’ by CART analysis (Bio-Rad Laboratories) [ ].

Techniques: Infection, Control, Biomarker Discovery

Journal: PLoS ONE

Article Title: Identification of Candidate Serum Biomarkers for Schistosoma mansoni Infected Mice Using Multiple Proteomic Platforms

doi: 10.1371/journal.pone.0154465

Figure Lengend Snippet: A nonparametric procedure, was used to generate candidate diagnostic algorithms. The CART based on a series of binary decision trees that recursively partition a data set into blocks of predicted positive and negative samples. The CART procedure pursues to minimize a cost function that balances prediction errors in false-positive or false-negative results as well as the total number of biomarkers used. An example of decision tree classification using infected (G1) vs controls is shown. In this algorithm, the intensities of the 46106 Da biomarker establish the splitting rules. The samples have an intensity of ≤0.423 are placed in the left daughter node, and samples that have an intensity of ≥0.423 go to the right daughter node. Terminal red boxes = uninfected; blue boxes = acute infection.

Article Snippet: Biomarker Patterns SoftwareTM algorithms (Bio-Rad) were then used to analyze the peak intensities and to generate classification ‘trees’ by CART analysis (Bio-Rad Laboratories) [ ].

Techniques: Diagnostic Assay, Infection, Biomarker Discovery

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A and B) Cell-ECM adhesion was assessed in MEFs [wild-type (WT), CD13KO, and WT treated with CD13-blocking Ab SL13; (A)] and human epithelial [C33A cells expressing empty vector (EV), human CD13 (HCD13) or a phospho-tyrosine mutant CD13 (Y6F); (B)] plated in the presence of fibronectin (FN) for the indicated time periods. Cells attached to ECM were stained with crystal violet dye, and absorbance was measured at 595 nm. (C and D) Cell spreading was assessed in MEFs (C) and C33A cells (D) plated on FN and left alone to spread for the indicated time periods. Cells were then stained with antibody against paxillin and imaged using confocal microscopy. Spreading was measured in cells of similar nuclear size as the area circumscribed by paxillin staining (total cell body) using Fiji software. (E and F) Cell migration was assessed using an in vitro scratch assay, in which monolayers of MEFs (E) or C33As (F) grown on FN for 6 hours were allowed to migrate into the cleared space. For MEFs, the number of cells migrating into the wound area was counted; whereas for C33A cells, the distance traveled by the leading front was measured by a phase-contrast microscopy and Fiji software. Data are mean ± SD of 3 independent experiments. **P<0.01 and *P<0.05 by two-tailed student’s t test.

Article Snippet: Cell culture Human cervical cancer epithelial cells, C33A (passage 5–15) purchased from ATCC, were maintained in DMEM supplemented with 10% FBS, 1% P/S at 37°C and 5% CO 2 .

Techniques: Blocking Assay, Expressing, Plasmid Preparation, Mutagenesis, Staining, Confocal Microscopy, Software, Migration, In Vitro, Wound Healing Assay, Microscopy, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) C33A cells expressing EV, HCD13 or Y6F were treated with vehicle (Veh) or aluminum fluoride (AlF; 50 μM) for 30 min, and membrane fractions were purified from total cell lysates. Active ARF6-GTP in the fractions was measured by pull down assay and normalized to total ARF6. Validation of the membrane fraction was verified by expression of plasma membrane marker E-cadherin. (B) Plots depict quantification of immunoblot analysis of ARF6-GTP, CD13, and IQGAP1 normalized to E-cadherin in the plasma membrane. Data are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 by two-tailed student’s t test.

Article Snippet: Cell culture Human cervical cancer epithelial cells, C33A (passage 5–15) purchased from ATCC, were maintained in DMEM supplemented with 10% FBS, 1% P/S at 37°C and 5% CO 2 .

Techniques: Expressing, Membrane, Purification, Pull Down Assay, Biomarker Discovery, Clinical Proteomics, Marker, Western Blot, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) In a pulse-chase assay human β1-integrin was labeled with Ab clone 12G10 and expression of β1-integrin (green) was tracked in antibody-pulsed cells for 1 hour (P/1h) followed by chase in antibody free medium for 2 and 4 hours (C/2h or C/4h) by immunofluorescence analysis. Cells were counterstained with DAPI (blue), fixed, and imaged by confocal microcopy. Scale bar; 5μm. (B) Quantification of fluorescence intensity of 12G10 staining shown in (A) using Fiji software. (C) β1-integrin receptor surface recycling was measured in a pulse-chase assay with flow cytometric analysis of human β1-integrin Ab clone 12G10 in C33A cells expressing EV, HCD13 or Y6F in the presence or absence of recycling inhibitor primaquine (PQ, 1μ)M. D. Equivalent level of CD13 expression in C33A expressing phospho-tyrosine mutant Y6F compared to HCD13 indicated by Western Blot analysis. Images (A) are representative and data (B to D) are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 by two-tailed student’s t test.

Article Snippet: Cell culture Human cervical cancer epithelial cells, C33A (passage 5–15) purchased from ATCC, were maintained in DMEM supplemented with 10% FBS, 1% P/S at 37°C and 5% CO 2 .

Techniques: Pulse Chase, Labeling, Expressing, Immunofluorescence, Fluorescence, Staining, Software, Mutagenesis, Western Blot, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) Cells were grown on FN for 0–60 min and cells were fixed and stained for β1-integrin (9EG7; green) and one of three endosomal markers (red): Rab5 (early endosomal marker), Rab7 (late endosomal/lysosomal marker) and Rab11 (recycling endosomal marker). Cells were counterstained with DAPI (blue). Scale bar; 5μm. Blots are representative of 3 independent experiments. (B) Images represented in (A) were quantified by MetaMorph software and correlation co-efficient (r) of β1-integrin (9EG7) with endosomal markers determined by Pearson’s analysis are shown. *P<0.05 by two-tailed student’s t test. (C) Quantification of Rab5+, Rab7+ or Rab11+ puncta per cell for in WT and CD13KO MEFs. All cells in a field were counted and five fields were counted for each genotype. (D) C33A cells expressing EV and HCD13 were treated with cycloheximide (CHX; 100 μg/ml) to inhibit new protein synthesis for the indicated time. Cell lysates were analyzed by immunoblotting for β1-integrin using the monoclonal antibody 12G10. GAPDH served as a loading control. Blots are representative of 3 independent experiments. (E and F) Quantification of immunoblot analysis represented in (D) by Image J Pro as a ratio of 12G10 to GAPDH or 12G10 normalized to time=0h. Data are means +/− SD of 3 independent experiments. (G and H) Total RNA was extracted from C33A cells expressing EV or HCD13 treated with cycloheximide for the indicated time, and the abundance of ITGB1 (β1-integrin) and GAPDH mRNA was determined by RT-PCR run on ethidium bromide-stained gels. Gels were analyzed by Image J Pro, and the ratio was plotted. AU, arbitrary units. Data in (B, C, E, F, and H) are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 by two-tailed student’s t test.

Article Snippet: Cell culture Human cervical cancer epithelial cells, C33A (passage 5–15) purchased from ATCC, were maintained in DMEM supplemented with 10% FBS, 1% P/S at 37°C and 5% CO 2 .

Techniques: Staining, Marker, Software, Two Tailed Test, Expressing, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) Lysates from C33A cells expressing EV, HCD13 or Y6F were immunoprecipitated with biotinylated CD13 mAb or control IgG and probed for IQGAP1 and total ARF6 by immunoblot analysis. ARF6-GTP was detected by a pull-down assay of the immunoprecipitates using beads conjugated to the PBD binding domain of GGA3 for 1 hour at 4°C. (B) Lysates from T27N ARF6 dominant-negative mutant cells expressing EV, HCD13 or Y6F were immunoprecipitated with biotinylated CD13 mAb or control IgG and probed for IQGAP1, ARF6 or HA-tag by immunoblot analysis. Active ARF6 was detected by pull down with GGA3-conjugated beads from the immunoprecipitates. (C and D) ARF6 activity was measured in C33A cells expressing EV or HCDi3 after cell-ECM adhesion over the indicated time. Using ARF6 protein-binding domain (PBD) of the effector protein GGA3 conjugated beads, which specifically binds the GTP-bound form of ARF6, the subsequent pull-down of ARF6-GTP was quantified by immunoblot analysis using the ARF6-specific antibody. Blots (C) are representative and quantified data (D) are means ± SD of 3 independent experiments. (E and F) β1-integrin receptor recycling was measured in a pulse-chase assay with β1 integrin Ab clone 12G10 (for C33A cells; E) or 9EG7 (for MEFs; F) in the presence of N-myristolated ARF6 inhibitor peptide. Serum-starved cells were treated with β1-integrin Ab at 4°C for 30 min, pulsed for 1 hour to induce internalization, acid-stripped, washed, treated with myr-ARF6 peptide (10 μM) for 30 min, and allowed to recycle at 37°C for 2 to 4 hours. Paraformaldehyde-fixed cells were stained with fluorescently conjugated secondary Ab and MFI (Mean Fluorescence Intensity) of surface β1-integrin was analyzed by flow cytometry. Data are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.0i by two-tailed student’s t test.

Article Snippet: Cell culture Human cervical cancer epithelial cells, C33A (passage 5–15) purchased from ATCC, were maintained in DMEM supplemented with 10% FBS, 1% P/S at 37°C and 5% CO 2 .

Techniques: Expressing, Immunoprecipitation, Control, Western Blot, Pull Down Assay, Binding Assay, Dominant Negative Mutation, Activity Assay, Protein Binding, Pulse Chase, Staining, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A and B) In a scratch assay, following injury on the monolayer by creating a scratch, C33A cells expressing HCD13 were allowed to migrate to the wound and fixed with 4% paraformaldehyde at the 6-hour time point. Cells were stained with phalloidin (red; left rows) or CD13 (red; right rows) and IQGAP1 (green) and imaged using confocal microcopy; magnified inset of CD13/IQGAP1-stained C33A-HCD13 cells is shown (B). Scale bar; 5μm. DAPI (blue). (C) Quantification of the area of F-actin and IQGAP1 accumulation at the migrating front of the cell, represented in (A), normalized to total cell area by Fiji software. Five fields were counted for each genotype, and all cells in each field were measured. (D) Percent of F-actin+, CD13+ and IQGAP1+ cells from (A) at the leading front were measured in each of five fields for each genotype. Data are mean ± SD of 3 independent experiments. *P<0.05 by two-tailed student’s t test.

Article Snippet: Cell culture Human cervical cancer epithelial cells, C33A (passage 5–15) purchased from ATCC, were maintained in DMEM supplemented with 10% FBS, 1% P/S at 37°C and 5% CO 2 .

Techniques: Wound Healing Assay, Expressing, Staining, Software, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) Immunoblot analysis of lysates from C33A cells expressing EV, HCD13 or Y6F, immunoprecipitated (i.p.) with CD13 mAb or control IgG, and probed (IB) for EFA6 and CD13 (A) or ARNO and CD13 (B). (C and D) Representative images (C) and quantitative analysis (D) of the number (per field assessed) of EFA6+ puncta (green) at the leading edge of cells expressing EV, HCD13 (red, top), or Y6F (red, bottom). Scale bar; 5μm. Data are mean ± SD of 3 independent experiments. *P<0.05 by two-tailed student’s t test.

Article Snippet: Cell culture Human cervical cancer epithelial cells, C33A (passage 5–15) purchased from ATCC, were maintained in DMEM supplemented with 10% FBS, 1% P/S at 37°C and 5% CO 2 .

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Two Tailed Test