Journal: Science Advances

Article Title: Tumor collection/processing under physioxia uncovers highly relevant signaling networks and drug sensitivity

doi: 10.1126/sciadv.abh3375

Figure Lengend Snippet: Cells for flow cytometry analysis were cultured for 5 days, and only lineage-negative (CD31-PE, CD45-PE, and CD140b-PE negative) cells were gated for the analysis. ( A ) CD49f/EpCAM staining patterns ( n = 9). ( B ) The number of CD49f + /EpCAM − cells varied on the basis of O 2 tension and tumor type. ( C ) EpCAM positivity also showed tumor-specific variability under physioxia compared to ambient air. ( D ) CD24/CD44 staining patterns of cells from ascites fluid under physioxia and ambient air ( n = 9). ( E ) Percentage of CD44 + cells in each sample under physioxia and ambient air. ( F ) Percentage of CD24 + cells under physioxia and ambient air. CD24 + cells were lower under physioxia compared to ambient air. ( G ) Cells from ascites fluids collected under physioxia contained elevated levels of BRD4 protein compared to cells collected under ambient air.

Article Snippet: The antibodies used against mouse cells were CD31-PE/Cyanine7 (A14715) from Molecular Probes; CD45-PE/Cyanine7 (25-0451-82), EpCAM-APC (allophycocyanin) (17-5791-82), and CD29-FITC (fluorescein isothiocyanate) (11-0291-82) from Invitrogen; CD140a-PE/Cyanine7 (323508) from BioLegend; LGR5-PE (phycoerythrin) (FAB8240P), TSPAN8-APC (FAB6524A), and CD49f-PE (FAB13501P) from R&D Systems; and CD61-FITC (561911), CD274-PE (558091), CD24-APC (562349), and CXCR4-FITC (551967) from BD Pharmingen; isotype control antibodies used included PE/Cyanine7 (400522) and APC (400612) from BioLegend and PE (554689) and FITC (553971) from BD Pharmingen.

Techniques: Flow Cytometry, Cell Culture, Staining

Journal: Cancer Research

Article Title: Survivin Enhances Motility of Melanoma Cells by Supporting Akt Activation and α5 Integrin Upregulation

doi: 10.1158/0008-5472.can-10-0194

Figure Lengend Snippet: Figure 4. Survivin upregulates the α5 integrin, which mediates enhanced melanocyte migration on fibronectin. A, melanocytes 48 hours after infection with pAd-GFP or pAd-Surv were subjected to Western blotting for the indicated integrins, and actin as a loading control. Blots for α6 and β4 integrins were performed, but expression of these integrins was not detected (not shown). B, melanocytes were infected with pAd-GFP or pAd-Surv, then cell lysates were prepared at the indicated time points and analyzed by Western blotting for survivin, the α5 or β1 integrin, or actin. C, metastatic and primary melanoma cell lines were infected with pAd-GFP or pAd-Surv, then 48 hours later cell lysates were subjected to Western blotting. D, melanocytes were infected with pAd-GFP or pAd-Surv for 24 hours, then added to chambers (5 × 104 per well) with fibronectin (FBN)- or laminin (LAM)-coated inserts in the presence of 10 μg/mL of either control (IgG, α6) or α5-blocking (α5, α5β1) antibodies as indicated (Block Ab). After incubation for 48 hours, migrating cells were quantified (top left, top right). Errors bars, SE of triplicate determinations. *, P < 0.0001; **, P = 0.002. LOX cells were infected with either pAd-GFP or pAd-Surv, then after 24 hours a linear scratch was made using a pipette tip (indicated between solid lines) and indicated antibodies were added for 3 hours. Photographs were taken 24 hours later (bottom).

Article Snippet: For antibody blocking experiments, cells were reded in serum-freemedia containing 10 μg/mL antibody t α5 integrin (IIA1, BD Biosciences), α5β1 integrin , Covance), α6 integrin (MAB13501, R&D Systems), or protei cence r Res; 70(20) October 15, 2010 Research. on March 20, 201cancerres.aacrjournals.org Downloaded from l mouse IgG (Sigma), and then seeded onto the upper e of the transwell chamber.

Techniques: Migration, Infection, Western Blot, Control, Expressing, Blocking Assay, Incubation, Transferring

Journal: Cancer Research

Article Title: Survivin Enhances Motility of Melanoma Cells by Supporting Akt Activation and α5 Integrin Upregulation

doi: 10.1158/0008-5472.can-10-0194

Figure Lengend Snippet: Figure 5. The α5 integrin is required for survivin-enhanced melanoma cell migration. A, YU2 cells were infected with pAd-GFP or pAd-Surv, then 24 hours later cells were transfected with scrambled or α5 integrin-specific siRNA. Cells were lysed for Western analysis after 72 hours (left), or 24 hours after transfection were added (5 × 104 per well) to chambers with fibronectin-coated inserts and migrating cells were quantitated 48 hours later (right). Errors bars, SE of triplicate determinations. *, P < 0.001. B, LOX cells (1 × 104 per well) were treated and analyzed as in A. *, P < 0.001. C, YU2 (left) and LOX (right) cells were infected with pAd-GFP or pAd-Surv, then 24 hours later cells were transfected with scrambled or α5 integrin–specific siRNA. After 24 hours, cells were added (YU2, 5 × 104 per well; LOX, 1 × 104 per well) to Matrigel-coated inserts and invading cells were quantitated 48 hours later. Errors bars, SE of triplicate determinations. ns, not significant. D, survivin-mediated upregulation of α5 integrin requires activation of Akt but not Erk. LOX cells were infected with pAd-GFP or pAd-Surv, then 24 hours later treated with diluent, 6 μmol/L PI3K inhibitor wortmanin (left), or 20 μmol/L Erk inhibitor u0126 (right). After 24 hours, cell lysates were prepared for Western blotting.

Article Snippet: For antibody blocking experiments, cells were reded in serum-freemedia containing 10 μg/mL antibody t α5 integrin (IIA1, BD Biosciences), α5β1 integrin , Covance), α6 integrin (MAB13501, R&D Systems), or protei cence r Res; 70(20) October 15, 2010 Research. on March 20, 201cancerres.aacrjournals.org Downloaded from l mouse IgG (Sigma), and then seeded onto the upper e of the transwell chamber.

Techniques: Migration, Infection, Transfection, Western Blot, Activation Assay