Journal: PLoS ONE

Article Title: Regeneration of esophagus using a scaffold-free biomimetic structure created with bio-three-dimensional printing

doi: 10.1371/journal.pone.0211339

Figure Lengend Snippet: The bio-3D printed structures are shown before transplantation. A-D: HE staining. E-H: Immunohistochemical staining with anti-αSMA antibodies. I-L: Immunohistochemical staining with anti-VEGF antibodies. M-P: immuno-histochemical staining with anti-CD31 antibodies. Q-T: Immunohistochemical staining with anti-elastin antibodies. Scale bar = 100 μm. HE: hematoxylin-eosin, SMA: smooth muscle actin, VEGF: vascular endothelial growth factor.

Article Snippet: Immunohistochemistry was performed with the following primary antibodies: alpha smooth muscle antibody (αSMA; 1:200; mouse monoclonal; A5228; SIGMA-ALDRICH, St. Louis, MO, USA) for evaluation of the smooth muscle cell distribution, anti-CD31 (1:400; rabbit polyclonal; bs-195R; Bioss, Boston, MA, USA) for evaluation of the endothelial cell distribution, anti-vascular endothelial growth factor (VEGF; 1:200; rabbit polyclonal; sc-152; Santa Cruz, Texas, USA) for evaluation of vasculogenesis, elastin (1:200; rabbit polyclonal; ab21610; Abcam, Cambridge, UK) for evaluation of generation of ECM, anti-pan-cytokeratin (1:200, mouse monoclonal; ab7753; Abcam) for evaluation of epithelialization, and anti-HLA class I ABC antibody (1:20, mouse monoclonal; ab70328; Abcam) for evaluation of distribution of implanted structure made of human cells.

Techniques: Transplantation Assay, Staining, Immunohistochemical staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Merkel Cell Carcinoma Expresses Vasculogenic Mimicry: Demonstration in Patients and Experimental Manipulation in Xenografts

doi: 10.1038/labinvest.2014.99

Figure Lengend Snippet: Biomarkers employed for identification of VM versus true angiogenesis.

Article Snippet: All sections were incubated overnight with primary antibodies at room temperature (~25°C) followed by 2-hour incubation with HRP-conjugated goat anti-rabbit (for Ulex, human and mouse CD31, CD144 and laminin), horse anti-goat (for VEGFR-1) and horse anti-mouse (for Nodal) secondary antibodies (all Vector Laboratories, Burlingame, CA) at room temperature.

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Merkel Cell Carcinoma Expresses Vasculogenic Mimicry: Demonstration in Patients and Experimental Manipulation in Xenografts

doi: 10.1038/labinvest.2014.99

Figure Lengend Snippet: WaGa and MKL-1-derived xenograft tumors contained CD31 − anastomosing networks that were enhanced with PAS staining, and were associated with staining for laminin (Lam), CD144, Nodal and VEGFR-1. Note the density and complexity of laminin + structures in comparison to CD31 + vessels (CD31 and Lam panels original magnification, 200×; PAS, CD144, Nodal, and VEGFR-1 original magnification, 1000×).

Article Snippet: All sections were incubated overnight with primary antibodies at room temperature (~25°C) followed by 2-hour incubation with HRP-conjugated goat anti-rabbit (for Ulex, human and mouse CD31, CD144 and laminin), horse anti-goat (for VEGFR-1) and horse anti-mouse (for Nodal) secondary antibodies (all Vector Laboratories, Burlingame, CA) at room temperature.

Techniques: Derivative Assay, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Merkel Cell Carcinoma Expresses Vasculogenic Mimicry: Demonstration in Patients and Experimental Manipulation in Xenografts

doi: 10.1038/labinvest.2014.99

Figure Lengend Snippet: MKL-1 tumors treated with vehicle, carboplatin and etoposide stained for CD31 (upper panels; original magnification, 100×) and laminin (Lam) (middle panels; original magnification, 100×); rectangular regions are representative fields enlarged for clarity (original magnification, 1000×). Note the marked and widespread increase in laminin + networks after chemotherapy. A similar picture is observed in WaGa derived tumors, although less diffusely (data not shown). A statistically significant increase in laminin immunoreactivity after carboplatin is observed in both cell lines, and after etoposide in MKL-1 derived tumors (graphs, lower panels).

Article Snippet: All sections were incubated overnight with primary antibodies at room temperature (~25°C) followed by 2-hour incubation with HRP-conjugated goat anti-rabbit (for Ulex, human and mouse CD31, CD144 and laminin), horse anti-goat (for VEGFR-1) and horse anti-mouse (for Nodal) secondary antibodies (all Vector Laboratories, Burlingame, CA) at room temperature.

Techniques: Staining, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Merkel Cell Carcinoma Expresses Vasculogenic Mimicry: Demonstration in Patients and Experimental Manipulation in Xenografts

doi: 10.1038/labinvest.2014.99

Figure Lengend Snippet: Relative LAMA3 (left), LAMB3 (center, left), LAMC2 (center, right) and CD144 (right) mRNA expression (mean ± s.e.m.) by carboplatin- and etoposide-resistant versus vehicle-treated MKL-1 (top) and WaGa (bottom) cells, as determined by real-time quantitative RT-PCR. Established human umbilical vein endothelial cells (HUVEC) served as a positive control. Data are representative of n=3 independent experiments. P<0.05, ** P<0.01, *** P<0.001).

Article Snippet: All sections were incubated overnight with primary antibodies at room temperature (~25°C) followed by 2-hour incubation with HRP-conjugated goat anti-rabbit (for Ulex, human and mouse CD31, CD144 and laminin), horse anti-goat (for VEGFR-1) and horse anti-mouse (for Nodal) secondary antibodies (all Vector Laboratories, Burlingame, CA) at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Positive Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Merkel Cell Carcinoma Expresses Vasculogenic Mimicry: Demonstration in Patients and Experimental Manipulation in Xenografts

doi: 10.1038/labinvest.2014.99

Figure Lengend Snippet: PAS-positive networks also are demonstrable by laminin IHC, and a minority of these are associated with CD31 staining in adjacent sections original magnification, 100×; inset, 1000×). Double IF labeling for CD31 and CD144 demonstrates an architectural pattern of CD144 positivity similar to that seen with laminin, and distinct from the comparatively few and discrete CD31 + vessels (original magnification, 200×)

Article Snippet: All sections were incubated overnight with primary antibodies at room temperature (~25°C) followed by 2-hour incubation with HRP-conjugated goat anti-rabbit (for Ulex, human and mouse CD31, CD144 and laminin), horse anti-goat (for VEGFR-1) and horse anti-mouse (for Nodal) secondary antibodies (all Vector Laboratories, Burlingame, CA) at room temperature.

Techniques: Staining, Labeling

Journal: bioRxiv

Article Title: An epigenetic switch regulates the ontogeny of AXL-positive/EGFR-TKI resistant cells by modulating miR-335 expression

doi: 10.1101/2021.01.06.425631

Figure Lengend Snippet: A) Immunoblot analysis of AXL in AXL-positive cells (H1650-M3 and PC14) and AXL −ve cells (H1650 and PC9). α-Tubulin is used as a loading control. B) Flow cytometry based analysis of surface expression of AXL in the AXL-positive cells lines (H1650-M3 and PC14) and AXL −ve cells (H1650 and PC9). Monoclonal antibody against N-terminal of AXL was used for the FACS analysis. Isotype control was used for identifying AXL-negative population. C) The chart represents the percentage of AXL-positive cells present in Erlotinib resistant and Erlotinib naïve cell lines. Erlotinib resistant cell lines are indicated in red, Erlotinib naïve EGFR mutant cell lines are indicated in blue, and Erlotinib naïve EGFR WT cell lines are indicated in green. Each bar represents mean ± SD of 3 replicates from two independent experiments. D) The chart represents relative AXL mRNA expression in the indicated cell lines or cells sorted on the basis of surface expression of AXL. Expression in AXL-positive cells was calculated relative to its expression in AXL-negative control cells. mRNA expression was quantified by SYBR-green-based RT-qPCR. Each bar represents mean ± SD of 3 replicates from two independent experiments. (p-value *** < 0.0005, **** < 0.00005, unpaired t-test). E) The chart represents relative Gas6 mRNA expression in the indicated cell lines or cells sorted based on surface expression of AXL. Expression in AXL-positive cells was calculated relative to its expression in AXL-negative control cells. mRNA expression was quantified by SYBR-green-based RT-qPCR. Each bar represents mean ± SD of 3 replicates from two independent experiments. (p-value *< 0.05, ** < 0.005, unpaired t-test). F) On the left panel, immunoblot analysis of AXL, GAS6, p120 RASGAP (loading control), p-AKT and AKT in AXL −ve (PC9) and AXL-positive (PC14) cells. On the right, cell extracts were immunoprecipitated with anti-AXL antibody and immunoblotted with phospho-tyrosine and AXL antibodies. Antibody heavy chain is shown as a loading control for the immunoprecipitation. G) The chart represents percentage of AXL-positive cells in six NSCLC patient tumors. Tumor-derived single cell suspension was stained with antibodies against CD45, CD31, EpCAM, and AXL. CD45-; CD31-; EpCAM+ cells were then FACS sorted for the AXL-positive populations. Each bar represents mean ± SD of three technical replicates. 20,000 cells were analyzed by FACS for each replicate of each sample. Schematic of the FACS sorting is presented in Figure 1-figure supplement 2. H) Expression of AXL and GAS6 genes in FACS-sorted AXL-negative (Blue) and AXL-positive (Red) cells from five human primary NSCLC tumors. mRNA expression was quantified by Cells to CT one step SYBR-green-based RT-qPCR. Expression of an indicated mRNA in the AXL-positive cells was calculated relative to its expression in AXL-negative cells from the respective tumor. Each dot represents mean ± SD of three replicates.

Article Snippet: APC anti-human AXL antibody (R & D Systems); cat # FAB154A Alexa Fluor 488 anti-human AXL PE-CF594 anti-human CD45 antibody (BD Biosciences); cat. # 562279 BV421 anti-human CD31 antibody (BD Biosciences); cat # 564089 Alexa Fluor 488 anti-human CD326 (Ep-CAM) antibody (BioLegend); cat # 324210

Techniques: Western Blot, Flow Cytometry, Expressing, Mutagenesis, Negative Control, SYBR Green Assay, Quantitative RT-PCR, Immunoprecipitation, Derivative Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: HIV-1 Tat Protein Enters Dysfunctional Endothelial Cells via Integrins and Renders Them Permissive to Virus Replication

doi: 10.3390/ijms22010317

Figure Lengend Snippet: Blockade of Tat entry in IC-HUVEC by antibodies directed against the α5β1, αvβ3, and/or αvβ5 integrin. In ( A ), activated HUVEC were suspended and then incubated for 2 h with 5 μg/mL of monoclonal antibodies (mAb) directed against CD31 (upper left panel), α5β1 (upper right panel), or αvβ3 (lower left panel), or with anti-α5β1 and anti-αvβ3 mAb, combined together at 5 μg/mL each (lower right panel). In ( B ), IC-HUVEC were suspended and then incubated for 2 h with 5 μg/mL of anti-α6β4 mAb (upper left panel), or with 5 μg/mL of anti-αvβ5 mAb, alone (upper right panel) or combined with anti-α5β1 and anti-αvβ3 mAb (lower panel). For both ( A , B ), IC-HUVEC incubated with Ab dilution buffer (PBS-0.1% BSA) were employed as controls (Nil). After incubation, IC-HUVEC was exposed for 10 min to biologically active Tat (1–1000 ng/mL) or its buffer. Intracellular Tat content was assayed by intracellular staining and flow cytometry as described. Results are expressed as the percentage of positive cells as compared to isotype-stained samples and non-permeabilized cells. Box-plot data obtained from three to four independent experiments and analyzed by the Mann–Whitney test are shown. Dots indicate individual measures. Orange plots refer to IC-HUVEC incubated with the said mAbs; blue plots refer to control IC-HUVEC.

Article Snippet: The anti-CD31 (PECAM-1) mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Bioprocessing, Staining, Flow Cytometry, MANN-WHITNEY, Control