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

Journal: Cancer Science

Article Title: C644‐0303, a small‐molecule inhibitor of the Wnt/β‐catenin pathway, suppresses colorectal cancer growth

doi: 10.1111/cas.15118

Figure Lengend Snippet: C644‐0303 was identified as an inhibitor of Wnt/β‐catenin signaling by a TCF/LEF‐based luciferase drug screening system. A, HCT‐116‐Luc cells were treated with 18 840 drugs (20 μmol/L) respectively, and the luciferase activity was detected 24 h later. B, Flowchart of high‐throughput drug screening. C, Chemical structure and SMILES of C644‐0303. The effects of C644‐0303 on TCF/LEF luciferase activity for 24 h (D), and cell viability for 72 h (E), in HCT‐116‐Luc cells were measured. F, Cell viability upon C644‐0303 treatment at the indicated concentrations for 72 h. G, The basal level of β‐catenin, active β‐catenin (Ser33/37/Thr41), and active β‐catenin (Ser45) in the indicated cells were analyzed by western blot. H, HEK293T cells were treated with a control‐conditioned medium (Ctrl‐CM) or Wnt3a‐conditioned medium (Wnt3a‐CM) in the presence of DMSO or C644‐0303 (20 μmol/L) for 18 h, followed using western blot analysis. Levels of active β‐catenin (Ser45) (I), and c‐Myc (J), were quantified using ImageJ software. Wnt3a‐CM stimulated HEK293T cells were treated with C644‐0303 (20 μmol/L) for 8 h. The mRNA levels of Cyclin D1 (K), and Axin2 (L) were detected using real‐time PCR. Error bars indicate means ± SEM. * P < .05, significant, one‐way ANOVA

Article Snippet: Antibodies against β‐catenin (D10A8), non‐phospho active β‐catenin (Ser45) (D2U8Y), non‐phospho (active) β‐catenin (Ser33/37/Thr41) (D13A1), phospho‐β‐catenin (Ser33/37/Thr41), phospho‐β‐catenin (Ser552) (D8E11), phospho‐β‐catenin (Ser675) (D2F1), GSK‐3β (3D10), phospho‐GSK‐3β (Ser9) (D85E12), Akt (pan) (C67E7), phospho‐Akt (Ser473) (D9E), Histone H3 (D1H2), cyclin D1, β‐actin (13E5), E‐cadherin (24E10), Axin2 (76G6), c‐Myc (D3N8F), N‐cadherin (D4R1H), Vimentin (D21H3), and Snail (C15D3) were from Cell Signaling Technology, α‐tubulin (B‐7) from Santa Cruz, and GAPDH from Kang Chen.

Techniques: Luciferase, Drug discovery, Activity Assay, High Throughput Screening Assay, Western Blot, Control, Software, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: C644‐0303, a small‐molecule inhibitor of the Wnt/β‐catenin pathway, suppresses colorectal cancer growth

doi: 10.1111/cas.15118

Figure Lengend Snippet: C644‐0303 inhibits β‐catenin activity and its downstream target gene expression. HCT‐116 cells were treated with C644‐0303 for 18 h. A, The cytoplasmic and nuclear protein were separated and analyzed using western blot. GAPDH and Histone H3 serve as a loading control of cytoplasmic and nuclear respectively. B, F, Whole‐cell lysates were analyzed using western blot. C‐E, HCT‐116 cells were treated with C644‐0303 (20 μmol/L) for 18 h. The mRNA levels of c‐Myc (C), Axin2 (D) and Cyclin D1 (E) were determined using real‐time PCR. G, HCT‐116 cells were treated with C644‐0303 for 24 h. The mRNA levels of MMP‐7 were determined using real‐time PCR. HT‐29 (H) and DLD‐1 (I) cells were treated with C644‐0303 for 18 h, whole‐cell lysates were analyzed using western blot. Error bars indicate means ± SEM. * P < .05, significant, Student t test for (C‐E), one‐way ANOVA for (G)

Article Snippet: Antibodies against β‐catenin (D10A8), non‐phospho active β‐catenin (Ser45) (D2U8Y), non‐phospho (active) β‐catenin (Ser33/37/Thr41) (D13A1), phospho‐β‐catenin (Ser33/37/Thr41), phospho‐β‐catenin (Ser552) (D8E11), phospho‐β‐catenin (Ser675) (D2F1), GSK‐3β (3D10), phospho‐GSK‐3β (Ser9) (D85E12), Akt (pan) (C67E7), phospho‐Akt (Ser473) (D9E), Histone H3 (D1H2), cyclin D1, β‐actin (13E5), E‐cadherin (24E10), Axin2 (76G6), c‐Myc (D3N8F), N‐cadherin (D4R1H), Vimentin (D21H3), and Snail (C15D3) were from Cell Signaling Technology, α‐tubulin (B‐7) from Santa Cruz, and GAPDH from Kang Chen.

Techniques: Activity Assay, Targeted Gene Expression, Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: C644‐0303, a small‐molecule inhibitor of the Wnt/β‐catenin pathway, suppresses colorectal cancer growth

doi: 10.1111/cas.15118

Figure Lengend Snippet: C644‐0303 potently inhibits TCF/LEF activity and modulates β‐catenin activity associated phosphorylation. A, Schematic diagram of Transcription Factor (TF) Activation Profiling Plate Array II. B, Nuclear extracts of HCT‐116 cells incubated with 20 μmol/L C644‐0303 for 18 h were processed as in (A). Scaled fold change of relative light units (RLU) of TFs in C644‐0303 vs vehicle treated cells was determined. C, Heatmap of the normalized RLU of Top20 TFs. D, Graphical summary of tumor‐associated functions of Top20 TFs using Ingenuity Pathways Analysis (IPA). Wnt3a‐CM stimulated HEK293T cells were treated with C644‐0303 (20 μmol/L) for 8 h in (E) and 18 h in (F), and analyzed using western blot

Article Snippet: Antibodies against β‐catenin (D10A8), non‐phospho active β‐catenin (Ser45) (D2U8Y), non‐phospho (active) β‐catenin (Ser33/37/Thr41) (D13A1), phospho‐β‐catenin (Ser33/37/Thr41), phospho‐β‐catenin (Ser552) (D8E11), phospho‐β‐catenin (Ser675) (D2F1), GSK‐3β (3D10), phospho‐GSK‐3β (Ser9) (D85E12), Akt (pan) (C67E7), phospho‐Akt (Ser473) (D9E), Histone H3 (D1H2), cyclin D1, β‐actin (13E5), E‐cadherin (24E10), Axin2 (76G6), c‐Myc (D3N8F), N‐cadherin (D4R1H), Vimentin (D21H3), and Snail (C15D3) were from Cell Signaling Technology, α‐tubulin (B‐7) from Santa Cruz, and GAPDH from Kang Chen.

Techniques: Activity Assay, Phospho-proteomics, Activation Assay, Incubation, Western Blot

Journal: Cancer Science

Article Title: C644‐0303, a small‐molecule inhibitor of the Wnt/β‐catenin pathway, suppresses colorectal cancer growth

doi: 10.1111/cas.15118

Figure Lengend Snippet: C644‐0303 inhibits the spheroidization and growth of CRC cells. Phase‐contrast images of HCT‐116 cells grown in embedded assay (A) or in on‐top assay (C) and treated as indicated. Scale bars represent 50 μm in ×200 and 200 μm in ×50 magnification. The black arrows mark the typical spheres. The spherical diameters were monitored, embedded (B) and on‐top (D). At the end of experiments, total protein of spheres embedded (E) and on‐top (F) was extracted and determined by BCA assay. (G) Whole‐cell lysates obtained in (E) were analyzed using western blot. (H) Active β‐catenin (Ser45) levels in (G) were quantified using ImageJ software and normalized to GAPDH levels. HCT‐116 cells were grown in embedded assay and treated with 10 μmol/L C644‐0303 for 5 d. The mRNA levels of Axin2 (I) and Cyclin D1 (J) were determined using real‐time PCR. HT‐29 cells were treated as in (B) and (D). At 6 d later, total protein of HT‐29 cell spheres was extracted and determined by BCA assay, embedded (K) and on‐top (L). HCT‐116 cells were treated with C644‐0303 (20 μmol/L, 18 h). The mRNA levels of LGR5 (M) and CD44 (N) were determined using real‐time PCR. Error bars indicate means ± SEM. * P < .05, significant, two‐way ANOVA for (B) and (D), one‐way ANOVA for (E), (F), (H), (K) and (L), Student t test for (I), (J), (M) and (N)

Article Snippet: Antibodies against β‐catenin (D10A8), non‐phospho active β‐catenin (Ser45) (D2U8Y), non‐phospho (active) β‐catenin (Ser33/37/Thr41) (D13A1), phospho‐β‐catenin (Ser33/37/Thr41), phospho‐β‐catenin (Ser552) (D8E11), phospho‐β‐catenin (Ser675) (D2F1), GSK‐3β (3D10), phospho‐GSK‐3β (Ser9) (D85E12), Akt (pan) (C67E7), phospho‐Akt (Ser473) (D9E), Histone H3 (D1H2), cyclin D1, β‐actin (13E5), E‐cadherin (24E10), Axin2 (76G6), c‐Myc (D3N8F), N‐cadherin (D4R1H), Vimentin (D21H3), and Snail (C15D3) were from Cell Signaling Technology, α‐tubulin (B‐7) from Santa Cruz, and GAPDH from Kang Chen.

Techniques: BIA-KA, Western Blot, Software, Real-time Polymerase Chain Reaction

Journal: Immunity

Article Title: Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual

doi: 10.1016/j.immuni.2019.02.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: L-a-Phosphatidylinositol-4-phosphate extracted from porcine brain (PIP) , Avanti Polar Lipids , Cat# 840045P.

Techniques: Purification, Recombinant, Staining, Plasmid Preparation, Luciferase, Cell Culture, Lysis, Reporter Gene Assay, Protease Inhibitor, Modification, Amplification, Binding Assay, Transfection, Crystallization Assay, Sequencing, Expressing, Software

Journal: Frontiers in Immunology

Article Title: Identification of Potent and Safe Antiviral Therapeutic Candidates Against SARS-CoV-2

doi: 10.3389/fimmu.2020.586572

Figure Lengend Snippet: High-throughput screening of US FDA-approved drug library to inhibit human coronavirus OC43 replication in vitro . (A) The strategy of high throughput screening to identify antiviral drugs that effectively inhibit OC43 replication. LLC-MK2 cells were pretreated with FDA-approved drugs at 10 μM for 1 h and infected with 1 MOI of OC43 for 48 h. Cells were then fixed and stained to calculate the infection ratio with Operetta software. (B) Primary screening results of 1,700 FDA-approved drugs against OC43, each dot represents one compound along with the rate of OC43 inhibition. (C) Image samples show signals of OC43 infection in cell cultures. LLC-MK2 cells were treated with indicated doses of remdesivir for 1 h, and then cells were infected with OC43 for 48 h. (D) Cell viability of remdesivir to LLC-MK2 cells were measured by CCK-8 assays. The % inhibitions were calculated according to the data in (C) .

Article Snippet: US FDA-approved drug library which contains 1,700 compounds were purchased from TargetMol (Massachusetts, USA).

Techniques: Sample Throughput, Screening, Drug discovery, In Vitro, Infection, Staining, Software, Inhibition, CCK-8 Assay