Journal: eLife

Article Title: Functionally heterogeneous human satellite cells identified by single cell RNA sequencing

doi: 10.7554/eLife.51576

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-Human CD45 (Beads) , Miltenyi Biotec , 130-045-801 , FACS.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, Software

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: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Expressing, Purification, Pull Down Assay, 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) 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: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Expressing, Immunoprecipitation, 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: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

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: In wild-type cells, phospho-CD13 and β1-integrin internalize into early endosomes, sort to recycling endosomes and return to the cell membrane, enabling cell-ECM adhesion and migration. However, in cells lacking CD13 or expressing an inactive CD13 mutant, whereas β1-integrin internalizes into early endosomes, it aberrantly traffics to Rab7+ lysosomes and it is ultimately degraded. Mechanistically, CD13 must be present in a complex containing the scaffolding protein IQGAP1, active-ARF6 its GEF EFA6 and β1-integrin at the plasma membrane to allow proper β1-integrin recycling and cell migration to proceed. In the absence of CD13, no active-ARF6 is detected in the plasma membrane and IQGAP1 is not recruited to the migrating front, thereby diminishing cell adhesion, spreading and migration.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Migration, Expressing, Mutagenesis, Scaffolding

Journal: medRxiv

Article Title: Identification and validation of heterotypic cell-in-cell structure as an adverse prognostic predictor for young patients of resectable pancreatic ductal adenocarcinoma

doi: 10.1101/2020.07.08.20148825

Figure Lengend Snippet: ( A ) Unprocessed composite core multiplex stained with E-cadherin, CD68, CD45, and DAPI. ( B ) Unmixed composite core pseudo-colored with yellow for E-cadherin, green for CD68, red for CD45, and blue for DAPI. ( C ) Spectral parameters for image unmixing. Fluorescent signals for different targets were captured based on the spectra indicated. ( D ) Images of single or merged channels displaying the boxed region in ( C ). Scale bar: 20 μm. ( E ) Representative images for the 4 CIC subtypes as indicated; scale bars: 5 μm;

Article Snippet: Samples were first stained with anti-CD45 antibody (mouse mAb from Boster, BM0091) at a dilution of 1:400 using Opal Multiplex tissue staining kit (Perkin Elmer, NEL791001KT) according to the manufacturer’s standard protocol.

Techniques: Multiplex Assay, Staining

Journal: medRxiv

Article Title: Identification and validation of heterotypic cell-in-cell structure as an adverse prognostic predictor for young patients of resectable pancreatic ductal adenocarcinoma

doi: 10.1101/2020.07.08.20148825

Figure Lengend Snippet: (A) CIC structures indicated by arrows in PDAC tissues stained by H&E. Boxed regions are zoomed in at the right. (B) CIC structures in PDAC tissues stained with antibodies for CD68 or CD45, respectively, by IHC. Boxed regions are zoomed in at the bottom. (C) Number of tissues positive in each CIC subtype. (D) The compositions of CIC subtypes in different TNM stages.

Article Snippet: Samples were first stained with anti-CD45 antibody (mouse mAb from Boster, BM0091) at a dilution of 1:400 using Opal Multiplex tissue staining kit (Perkin Elmer, NEL791001KT) according to the manufacturer’s standard protocol.

Techniques: Staining