Journal: The Journal of Cell Biology

Article Title: EMILIN1–α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation

doi: 10.1083/jcb.201008013

Figure Lengend Snippet: Lack of EMILIN1 up-regulates PI3K/Akt and Erk1/2 and down-regulates PTEN. (A) Representative Western blot analysis of 6–8-wk-old skin tissue extracts of WT and Emilin1 −/− mice. (B) Quantification of Western blot analysis reported in A by Quantity One software. The mean values (±SEM) of pSmad2 (Ser465/467 and Ser245/250/255), pErk1/2, PTEN, pAkt (Ser473), PI3K, Cyclin A, and Cdk2 relative expression levels of WT ( n = 8) and Emilin1 −/− ( n = 8) mice are reported. (C) Representative Western blot analysis of epidermis and dermis extracts of 7-wk-old WT and Emilin1 −/− mice. (A and C) Molecular mass is indicated in kilodaltons. (D) Quantification of Western blot analysis reported in C by Quantity One software. The mean values (±SD) of pSmad2 (Ser465/467 and Ser245/250/255), pErk1/2, PTEN, pAkt (Ser473), and PI3K relative expression levels of WT ( n = 4) and Emilin1 −/− ( n = 4) mice are reported. *, P = 0.05; **, P < 0.05; ***, P < 0.01.

Article Snippet: Moreover, mouse anti-CdK2 (BD), mouse anti–Cyclin D1/2 (Millipore), rabbit anti–mature TGF-β1 (BioVision Research Products), rabbit anti–pan-CK antibody (Dako), mouse anti–integrin α9β1 (clone Y9A2) and anti–integrin α4 (clone P1H4; Millipore), goat anti–mouse integrin α9β1 (LifeSpan BioSciences), and mouse anti–β1 integrin subunit (clone 4B4; Beckman Coulter) antibodies were used.

Techniques: Western Blot, Software, Expressing

Journal: Cell & Bioscience

Article Title: Subcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirus

doi: 10.1186/s13578-015-0066-2

Figure Lengend Snippet: Detection of KSHV in endosomal fractions. A Distribution of endosomal membranes was determined from uninfected and KSHV infected HFF cells using sucrose gradient fractions. Equal-volume aliquots from each gradient fraction were separated by SDS-PAGE and probed by Western blotting against organelle markers: Rab5 and EEA1 (EE markers), Rab7 (LE marker), LAMP1 (LE and lysosome marker), and histone H3 (nuclear marker). B Lysosome detection in sucrose gradient fractions of uninfected and KSHV infected HFF cells. Acid phosphatase activity was analyzed by using Acid Phosphatase Assay Kit (Sigma). C KSHV escapes from the LE. Following gradient centrifugation, viral genomic DNA was extracted from each recovered fraction. Fractions were then quantified for the content of internalized viral DNA by performing qPCR using specific primers. Data ( panels B , C ) represent the average ±SD ( error bars ) of three experiments. Columns with different alphabets indicate statistical significance (p < 0.05) by least significant difference (LSD)

Article Snippet: Anti-integrin α9 (H-198) antibodies (Santa Cruz Biotechnology); anti-integrin α5 (P1D6), β1 (6S6), α9β1 (Y9A2; Millipore) antibodies; Rab5 and Rab7 (D95F2) XP™ antibodies (Cell Signaling Technology); purified mouse anti-EEA1 (BD Transduction Laboratories) antibody; anti-LAMP1 antibody and Anti-Histone H3 antibody (Abcam); mouse monoclonal antibody (5B7B6) to KSHV orf62 encoded minor capsid protein, TRI-1 (Thermo Scientific) were used in this study.

Techniques: Infection, SDS Page, Western Blot, Marker, Activity Assay, Acid Phosphatase Assay, Gradient Centrifugation

Journal: Cell & Bioscience

Article Title: Subcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirus

doi: 10.1186/s13578-015-0066-2

Figure Lengend Snippet: Detection of KSHV in endosomal fractions. A Distribution of endosomal membranes was determined from uninfected and KSHV infected HFF cells using sucrose gradient fractions. Equal-volume aliquots from each gradient fraction were separated by SDS-PAGE and probed by Western blotting against organelle markers: Rab5 and EEA1 (EE markers), Rab7 (LE marker), LAMP1 (LE and lysosome marker), and histone H3 (nuclear marker). B Lysosome detection in sucrose gradient fractions of uninfected and KSHV infected HFF cells. Acid phosphatase activity was analyzed by using Acid Phosphatase Assay Kit (Sigma). C KSHV escapes from the LE. Following gradient centrifugation, viral genomic DNA was extracted from each recovered fraction. Fractions were then quantified for the content of internalized viral DNA by performing qPCR using specific primers. Data ( panels B , C ) represent the average ±SD ( error bars ) of three experiments. Columns with different alphabets indicate statistical significance (p < 0.05) by least significant difference (LSD)

Article Snippet: Anti-integrin α9 (H-198) antibodies (Santa Cruz Biotechnology); anti-integrin α5 (P1D6), β1 (6S6), α9β1 (Y9A2; Millipore) antibodies; Rab5 and Rab7 (D95F2) XPTM antibodies (Cell Signaling Technology); purified mouse anti-EEA1 (BD Transduction Laboratories) antibody; anti-LAMP1 antibody and Anti-Histone H3 antibody (Abcam); mouse monoclonal antibody (5B7B6) to KSHV orf62 encoded minor capsid protein, TRI-1 (Thermo Scientific) were used in this study.

Techniques: Infection, SDS Page, Western Blot, Marker, Activity Assay, Acid Phosphatase Assay, Gradient Centrifugation

Journal: Cell & Bioscience

Article Title: Subcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirus

doi: 10.1186/s13578-015-0066-2

Figure Lengend Snippet: Antibodies to integrin α9β1 inhibit KSHV escape from the late endosome. A KSHV is present in Rab5-positive EEs and Rab7-postive LEs. HFF cells were either uninfected or pre-treated (1 h at 37 °C) with antibodies (20 µg/ml) to α9β1 or pre-immune IgG prior to infection with FITC-KSHV for 1 min ( A ) and 30 min ( B ) at 37 °C. Cells were washed, fixed, and subsequently immunostained with TRITC labeled anti-Rab5 antibodies ( A ) and TRITC labeled anti-Rab7 antibodies ( B ) prior to mounting using an anti-fade reagent containing DAPI and examining under a fluorescent microscope. Representative images are provided at ×1000 magnification. The white circles indicate co-localization of FITC-KSHV with the respective endosome marker within the cell ( panels A and B )

Article Snippet: Anti-integrin α9 (H-198) antibodies (Santa Cruz Biotechnology); anti-integrin α5 (P1D6), β1 (6S6), α9β1 (Y9A2; Millipore) antibodies; Rab5 and Rab7 (D95F2) XPTM antibodies (Cell Signaling Technology); purified mouse anti-EEA1 (BD Transduction Laboratories) antibody; anti-LAMP1 antibody and Anti-Histone H3 antibody (Abcam); mouse monoclonal antibody (5B7B6) to KSHV orf62 encoded minor capsid protein, TRI-1 (Thermo Scientific) were used in this study.

Techniques: Infection, Labeling, Microscopy, Marker

Journal: Scientific Reports

Article Title: The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9β1 Affects Cancer Cell Behaviour

doi: 10.1038/srep28529

Figure Lengend Snippet: ( A ) G361 total cell lysates (TCLs) after adhesion to A12-Dis in the absence or presence of Mn 2+ (1 mM) were analysed by WB using an anti-phosphotyrosine (pTyr) antibody (4G10). Full activation of integrins by Mn 2+ leads to tyrosine phosphorylation of a 75-kDa (pp75) protein (arrowhead) compared to untreated cells. Total actin was used as loading control. ( B ) G361 cells in suspension with or without Mn 2+ (3 mM) blotted for pTyr (4G10) and actin. ( C ) Immunoprecipitation (IP) with anti-pTyr antibody (PY99) of TCL from G361 cells in suspension treated with Mn 2+ (3 mM). Samples were analysed by WB. TCL, preclear beads, antibody (Ab) alone, and mouse IgG (IgG), were included as controls. WBs were probed for pTyr (PY99), pY421 cortactin, and pY470 cortactin as indicated. ( D ) WB analysis of G361 TCL after 48 hours transfection with negative control (NC) or cortactin (CTTN) siRNA using cortactin antibody. ( E ) TCL from NC or CTTN siRNA treated cells was probed against pTyr (4G10) and actin. ( F ) Quantitation of ( E ) for relative levels of p75 tyrosine phosphorylation after Mn 2+ treatment. Data from Mn 2+ treated NC cells were set at 1.0. Data were analysed using unpaired two-tailed t-test.

Article Snippet: The following mouse monoclonal antibodies were used: anti-phosphotyrosine PY99 (Santa Cruz Biotechnology), anti-phosphotyrosine (4G10), anti-α9β1 antibody (Y9A2), anti-integrin α4 (HP2/1), anti-total β1 (MAB1959), anti-active β1 (12G10), anti-myc tag (4A6), anti-v-Src (OP07), anti-PTEN (6H2.1), anti-pan-actin (all from Millipore), activating β1-integrin antibody (TS2/16, Thermo Scientific), anti-Yes, anti-Fyn, anti-Hck, anti-Crk (all from BD Biosciences), anti-cellular/EDA fibronectin antibody (FN-3E2, Sigma).

Techniques: Activation Assay, Phospho-proteomics, Control, Suspension, Immunoprecipitation, Transfection, Negative Control, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Fibronectin-guided migration of carcinoma collectives

doi: 10.1038/ncomms14105

Figure Lengend Snippet: ( a ) Representative phase contrast images of HNSCC lines plated on plastic or TIF-derived ECM for 24 h. Scale bar, 150 μm. ( b ) Analysis of CAL33 cells on TIF-derived ECM by laser scanning confocal microscopy: (top) three-dimensional rendering of Dapi-stained nuclei and FN-EDA-stained ECM, (bottom) single projections of E-cadherin in intercellular junctions (left) and in tumour cell-derived vesicles sequestered in the ECM (right). Scale bar, 20 μm. ( c ) Migration of non-dividing CAL33 cells ( n ≥100) in cohorts was monitored by time-lapse videomicroscopy, 12 h after seeding on plastic or TIF-derived ECM. Images were acquired every 5 min for 24 h. Representative tracings (denoted by different grey levels) from origin of CAL33 cell cohort migration on plastic (No coat) or ECM are shown. ( d ) Tracks with different directionality ratios were simulated (mean square displacement (MSD) versus time), as described in Methods, and analysed in the same way as experimental data. ( e ) Histograms depict the velocity and directionality of cell movement on plastic (No coat) or ECM. For directionality, the confidence interval is measured as defined by MATLAB in the fit function (see ‘Analysis of cell migration' in Methods). Statistically significant data are indicated by * P <0.05, ** P <0.01, *** P <0.001 or **** P <0.0001. If no statistical difference, error bars are shown at 95%. Results from a representative experiment, of at least three, are shown.

Article Snippet: The following antibodies were used: polyclonal anti-Smad2, anti-phospho-Smad2 (Ser465/467), anti-Smad3 and anti-phospho-Smad3 (Ser423/425) from Cell Signalling Technology (Beverly, MA); monoclonal anti-FN and anti-E-cadherin from BD Biosciences (Le Pont de Claix, France); monoclonal anti-TNC (clone BC24), α-smooth muscle actin (clone 1A4), anti-β actin (clone AC-15) and polyclonal anti-periostin from Sigma-Aldrich; monoclonal anti-collagen I, blocking monoclonal α9β1 antibody (cloneY9A2), rabbit anti-pHis (Ser10) and anti-TGFBI from Abcam (Cambridge, MA); anti-FN-EDA (clone IST-9) from Sirius Biotech (Genoa, Italy); anti-ERK1 (clone C-16) from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-FN, monoclonal anti-αvβ3 integrin (clone LM609), monoclonal anti-α5β1 integrin (MAB1999), blocking monoclonal anti-α5β1 integrin (clone JBS5), monoclonal anti-αvβ5 integrin (clone P1F6), monoclonal anti-αvβ6 integrin (clone E7P6), monoclonal anti-collagen VI, anti-cortactin p80/85 and monoclonal anti-α9β1 integrin were from Millipore (Billerica, MA); anti-β1 integrin (clone lia1/2) from GenWay Biotech (San Diego, CA); blocking monoclonal anti-β1 (clone P5D2) from R&D systems; polyclonal anti-α9 integrin from Thermo Scientific (Rockford, IL); and mouse monoclonal anti-CD31 from DAKO Corp. (Carpinteria, CA).

Techniques: Derivative Assay, Confocal Microscopy, Staining, Migration