Journal: Oncology Reports

Article Title: Adipocytes induce the resistance of ovarian cancer to carboplatin through ANGPTL4

doi: 10.3892/or.2020.7647

Figure Lengend Snippet: Downstream effect of ANGPTL4 following binding to integrin α5β1 on surface of the tumor cell membrane. (A) Co-IP was performed and the immunoprecipitates were analyzed using western blot analysis with antibodies against integrin α5β1, claudin-5, ANGPTL4 and GAPDH. (B) Statistical results of the co-IP assay. The data are presented as the mean ± SD. **P<0.01 vs. SKOV3. (C and D) The regulatory effect following silencing of integrin α5β1 on the ANGPTL4-induced resistance of SKOV3 cells in vitro . The data are presented as the mean ± SD. **P<0.01 vs. SKOV3 shMock /CM/carboplatin or SKOV3 shMock /adipocyte/carboplatin). (E and F) The regulatory effect following silencing of claudin 5 on the ANGPTL4-induced resistance of SKOV3 cells in vitro . The data are presented as the mean ± SD. **P<0.01 vs. SKOV3 shMock /CM/carboplatin or SKOV3 shMock /adipocyte/carboplatin). ANGPTL4, angiopoietin-like 4; Co-IP, co-iummunoprecipitation; sh, short hairpin; CM, conditioned medium.

Article Snippet: Subsequently, the membranes were blocked with 5% skimmed milk, prepared with the TBS-Tween-20 buffer, and incubated with primary antibody against ANGPTL4 (1:1,000; cat. no. ab2920; Abcam), integrin α5β1 (1:1,000; cat. no. orb13515; Biorbyt Ltd.), claudin 5 (1:2,000; cat. no. ab131259; Abcam), VE-cadherin (1:1,000; cat. no. 2158; Cell Signaling Technology, Inc.), Bcl-xl (1:1,000; cat. no. ab32370; Abcam), ABCB1 (1:1,000; cat. no. 13342; Cell Signaling Technology, Inc.), ABCC1 (1:1,000; cat. no. 72202; Cell Signaling Technology, Inc.), ABCG2 (1:1,000; cat. no. ab207732; Abcam), β-actin (1:10,000; cat. no. BS6007M; Bioworld Technology, Inc.), PI3K (1:2,000; cat. no. 4257; Cell Signaling Technology, Inc.), phospho-PI3K (1:1,000; cat. no. 4228; Cell Signaling Technology, Inc.), c-Myc (1:2,000; cat. no. 18583; Cell Signaling Technology, Inc.), phospho-c-Myc (1:1,000; cat. no. 13748; Cell Signaling Technology, Inc.), p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), phospho-p65 (1:1,000; cat. no. ab194726; Abcam), Akt (1:2,000; cat. no. 4691; Cell Signaling Technology, Inc.), phospho-Akt (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.) overnight at 4°C, whilst avoiding non-specific background.

Techniques: Binding Assay, Membrane, Co-Immunoprecipitation Assay, Western Blot, In Vitro

Journal: Nature Communications

Article Title: Endothelial destabilization by angiopoietin-2 via integrin β1 activation

doi: 10.1038/ncomms6962

Figure Lengend Snippet: ( a – c ) HeLa cells seeded on fibronectin were stimulated with 4 μg ml −1 rhAng2 ( a , b ) or rhAng1 ( c ) for 30 min, or pretreated with β1-integrin-blocking antibody (4 μg ml −1 mab13, a , b ) or cilengitide (10 μM, b ) for 5 min, and then further stimulated for 30 min with rhAng2. The cells were stained for active β1-integrin (12G10) and for His-tag ( a , c ) or Ang2 ( b ). ( b ) Ang2-positive matrix adhesions were analysed from 7 (mAb13) or 10 (cilengitide) microscopic images/experiment (total of 500 cells/mAb13 treatment and 320 cells/cilengitide treatment, n =3 for mAb13, for cilengitide a representative experiment is shown, repeated three times) P =0.03 for mAb13 and P =0.496 for cilengitide, Student’s T -test. ( c ) Active β1-integrin was quantified from 7 microscopic images/experiment, total of 350 cells/treatment analysed, n =3, P =0.04 for control versus Ang2, P =0.01 for Ang2 versus Ang1, P =0.99 for control versus Ang1, Dunnet’s test. ( d ) CHO cells were incubated with fluorescently labelled fibronectin fragment (FN7–10) and with various concentrations of rhAng2 or rhAng1, as indicated. FN7–10 binding to CHO cells was quantified using fluorescence-activated cell sorting, and normalized to total α5β1 levels, as explained in the methods. P =0.0004 for 10 μg ml −1 (150 nM) ( n =3) and P =0.007 for 5 μg ml −1 (75 nM) ( n =3) concentration of rhAng2 versus FN7–10 only, Dunnet’s test. ( e ) CHO cells were treated with the indicated concentrations of rhAng1 or rhAng2, and integrin activation measured as in d . ( f ) Binding of Ang2–Flag to biotinylated β1-integrin ectodomain was measured in triplicate using ELISA, a representative experiment of two independent experiments is shown. ( g , h ) CHO cells transduced with a control plasmid, or plasmids encoding for flag-tagged Ang2, Ang1–Ang2 (Ang1–2) or Ang2–Ang1 (Ang2–1) chimeric proteins (schematic structures are shown in g ) were incubated with FN7–10 and, where indicated, with 10 μg ml −1 rhAng2. FN7–10 binding to CHO cells was quantified as in d . P =0.004 for rhAng2, P =0.002 for Ang2–Flag, P =0.001 for Ang2–1–Flag versus FN only, ( n =3, Dunnet’s test). Mean and s.d. * P <0.05, ** P <0.01, *** P <0.005. Nuclear Hoechst stain. Projections of confocal z-stacks. Scale bar, 20 μm.

Article Snippet: The following antibodies were used at dilution 1:100 for immunofluorescence (IF) staining, unless otherwise indicated: anti-hTie1 (AF619), anti-hTie2 (AF313, WB 1:4,000), anti-m/r-Tie2 (AF762, 1:1,000), anti-hAng2 (AF623) (R&D Systems), anti-mTie2/Tek4 (cat. 95–585, 1:80, Millipore, Billerica, MA), anti-hVE-cadherin (1:200, cat. 555661, Pharmingen, BD Biosciences; cat. 2500, Cell Signaling Technology, Danvers, MA), anti-mVE-cadherin (555289; BD Biosciences or 14–1441, eBioscience, San Diego, CA), anti-His-tag (1:40, 2365, Cell Signaling Technology), anti-Flag (1:1,000, F3648, M2, Sigma-Aldrich, St Louis, MO), anti-fibronectin (F3648, Sigma-Aldrich), anti-β1-integrin (1:30) , mab12G10 (1:300, Abcam, Cambridge, UK), mab2252 (Merck Millipore) and anti-hamster α5β1 (1:50, PB1, developed by Brown and Juliano and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa), anti-mouse CD31 (1:2,000, Mab1398Z, Millipore), anti-αvβ3 (1:40, mab1976, Millipore), Alexa-488, Alexa-594, Alexa-647-conjugated secondary antibodies (1:300, Life Technologies, Carlsbad, CA) and anti-hamster FITC (Jackson Immunoresearch, Westgrove, PA).

Techniques: Blocking Assay, Staining, Control, Incubation, Binding Assay, Fluorescence, FACS, Concentration Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation