Journal: Molecular Cancer Research

Article Title: The Opposing Effect of Hypoxia-Inducible Factor-2α on Expression of Telomerase Reverse Transcriptase

doi: 10.1158/1541-7786.mcr-07-0065

Figure Lengend Snippet: FIGURE 4. HIF-2a occupancy on the hTERT promoter, recruitment of p300, and changes in histone H3 acetylation in A498 and U251 cells under hypoxic conditions. A. Schematic presentation of the hTERT promoter and locations of the ChIP PCR amplicons relative to the translational start codon (ATG). The putative HREs in region a are indicated (16). Region b was also found to interact with HIF-1a according to Anderson et al. (10). B. The ChIP assay for the presence of HIF-2a, p300, and histone H3 acetylation at the hTERT promoter in hypoxia-treated A498 and U251 cells. The precipitated DNA was subject to PCR analysis using the primer pairs specific to the hTERT promoter regions a and b. Input is sonicated chromatin without immunoprecipitation, but reverse cross-linked and purified together with ChIP samples. HRE, hypoxia-responsive element; H and N, the cells were cultured under hypoxic and normal oxygen conditions, respectively. Ab, no antibody controls. H3-Ac, the antibody against acetylated histone H3.

Article Snippet: The membranes were probed with the specific mouse monoclonal antibody against HIF-1a or HIF-2a rabbit polyclonal antibody (Novus Biologicals), followed by a secondary anti-mouse or rabbit horseradish peroxidase–conjugated immunoglobulin G, and developed with the enhanced chemiluminescence method (Amersham).

Techniques: Sonication, Immunoprecipitation, Purification, Cell Culture

Journal: The Journal of biological chemistry

Article Title: Alzheimer disease-specific conformation of hyperphosphorylated paired helical filament-Tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation.

doi: 10.1074/jbc.M512786200

Figure Lengend Snippet: FIGURE 5. Anti-polyubiquitin staining of affinity-purified PHF-Tau. Following SDS- PAGE separation of MC1 affinity-purified PHF-Tau on a 10% polyacrylamide gel, Western blot was performed with an anti-polyubiquitin antibody. Immunoreactivity was visual- ized by enhanced chemiluminescence (left panel). Secondary antibody (goat anti-mouse IgG-horseradish peroxidase) control for nonspecific immunoreactivity is also depicted (right panel).

Article Snippet: Blots were incubated with a monoclonal mouse anti-polyubiquitin antibody (Novus Biologicals) 1:500 overnight at 4 °C.

Techniques: Staining, Affinity Purification, SDS Page, Western Blot, Control

Journal: The Journal of biological chemistry

Article Title: Alzheimer disease-specific conformation of hyperphosphorylated paired helical filament-Tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation.

doi: 10.1074/jbc.M512786200

Figure Lengend Snippet: FIGURE 6. Relative quantification of Lys-6, Lys-11, and Lys-48 polyubiquitin linkages by SRM. Polyubiquitin linkages at Lys-6 (A), Lys-11 (B), and Lys-48 (C) of ubiquitin (ubiquitinated and unmodified peptides) were quantified by specific precursor-to-product ion transition monitoring (Table 4). D shows chromatograms for all three ubiquitinated peptides on the same scale for visual comparison of the relative peak areas.

Article Snippet: Blots were incubated with a monoclonal mouse anti-polyubiquitin antibody (Novus Biologicals) 1:500 overnight at 4 °C.

Techniques: Quantitative Proteomics, Ubiquitin Proteomics, Comparison

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs.

doi: 10.1002/cyto.a.23847

Figure Lengend Snippet: Figure 1. Canine OSA cell lines OVC-cOSA-31 (A) and OVC-cOSA-75 (B), gated as indicated, were highly positive for intracellular Col I (center panel) and variably positive for OC (right panel). Blue = anti-rat CD4 control antibody; red = Col I or OC antibody. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The following isotype- and fluorochrome-matched antibodies were used as negative controls in all assays: (1) Mouse antirat CD4 monoclonal antibody OX-35 (IgG2a) conjugated to fluorescein isothiocyanate (FITC); (2) Mouse anti-rat CD71 monoclonal antibody OX-26 (IgG2a) conjugated to phycoerythrin (PE), both from BD (Mississauga, ON); (3) mouse monoclonal antibody to bovine retinal pigment epithelium (RPE) 65 (401.8B11.3D9, IgG1) conjugated to FITC (Novus Biologicals, Oakville, ON); and (4) mouse monoclonal antibody to human CD146 (P1H12, IgG1) conjugated to PE from EMD Millipore (Etobicoke, ON).

Techniques: Control

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs.

doi: 10.1002/cyto.a.23847

Figure Lengend Snippet: Figure 8. Cytomorphology of OSA cells. Cytocentrifuge preparations of (A) cultured OVC-cOSA-31 cells (400×); (B) normal dog blood leukocytes spiked with primary OSA tumor aspirate cells, fixed, and permeabilized (arrow on OSA cell, 400×); (C) CTC sorted based on Col I positivity from blood of dog with a primary OSA (1,000×); (D) cell directly aspirated from a primary OSA (1,000×) with neutrophil (arrow); (E) primary OSA cells aspirated from tumor and sorted based on Col I positivity (1,000×); (F) OVC-cOSA-31 cells sorted from spiked normal dog blood (1,000×). All slides were Wright stained. Immunofluorescence of Col I-FITC positive; (G) OVC-cOSA-31 cells; and (H) CTC cells sorted from blood of a dog with OSA. Nuclei are stained with DAPI (1,000×). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The following isotype- and fluorochrome-matched antibodies were used as negative controls in all assays: (1) Mouse antirat CD4 monoclonal antibody OX-35 (IgG2a) conjugated to fluorescein isothiocyanate (FITC); (2) Mouse anti-rat CD71 monoclonal antibody OX-26 (IgG2a) conjugated to phycoerythrin (PE), both from BD (Mississauga, ON); (3) mouse monoclonal antibody to bovine retinal pigment epithelium (RPE) 65 (401.8B11.3D9, IgG1) conjugated to FITC (Novus Biologicals, Oakville, ON); and (4) mouse monoclonal antibody to human CD146 (P1H12, IgG1) conjugated to PE from EMD Millipore (Etobicoke, ON).

Techniques: Cell Culture, Staining

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 1. LAT1 expression increases in pemetrexed-resistant NSCLC cells. (A) The primary or secondary tumorspheres were cultured from A549 and A400 cells according to the protocol described in the Materials and Methods section. Pictures

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Expressing, Cell Culture

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 2. Knockdown of LAT1 reduces CSC activity and Akt/mTOR activation in NSCLC cells. (A) The knockdown of LAT1 in A400 and H1299 cells was performed by lentiviral delivery of LAT1-specific shRNAs (shSLC7A5#1 or shSLC7A5#2) and 100 nM of LAT1-specific siRNA oligonucleotides, respectively, for 48 h. The CSC activity was determined by tumorsphere cultivation. The inserted scale bars indicate 100 µm in length. *** p< 0.001. (B) The mRNA expressions of cancer stemness genes (BMI1/SOX2/Oct4) and SLC7A5 were determined by real-time RT-PCR. * p < 0.05; ** p < 0.01; *** p < 0.001. (C) The protein expressions of phosphor-mTORser2481, mTOR, phosphor-Aktser473, Akt, and LAT1 were determined by Western blot analysis. GAPDH was used as a protein-loading control. The inserted numbers present relative expression folds in comparison to shLacZ or the control siRNA group.

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Knockdown, Activity Assay, Activation Assay, Quantitative RT-PCR, Western Blot, Control, Expressing, Comparison

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 3. Inhibition of LAT1 activity by JPH203 suppresses CSC activity and Akt/mTOR activation in NSCLC cells. (A) The CSC activity of A400 and H1299 cells was determined by tumorsphere cultivation, and pictures were presented at 100× magnification. The quantitative data were presented as relative percentages to the tumorsphere number of the 0.1% DMSO treated group (0 µM). The inserted scale bars indicate 100 µm in length * p < 0.05; ** p < 0.01. (B) The protein expressions of phosphor-mTORser2481, mTOR, phosphor-Aktser473, Akt, and LAT1 were determined by Western blot analysis. GAPDH was used as a protein-loading control. The inserted numbers presented are relative expression folds in comparison to the 0.1% DMSO treated group (0 µM) after normalization with the internal control of GAPDH. (C) The protein expression of BMI1 or SOX2 was determined by Western blot. The inserted numbers indicated relative expression levels as compared to DMSO group after normalization to the internal control of GAPDH. (D) The CSC activity of A400 cells under JPH203 treatment with or without 100 µM LLME cotreatment was determined by tumorsphere cultivation and pictures were presented at 200× magnification. The quantitative data were presented as relative percentages of the tumorsphere number of the 0.1% DMSO without LLME treatment group. The inserted scale bars indicate 100 µm in length. * p < 0.05; ** p < 0.01. (E) The protein expressions of phosphor-mTORser2481, mTOR, phosphor-Aktser473, Akt, and LAT1 in A400 and H1299 cells in the presence of LLME were determined by Western blot analysis. GAPDH was used as a protein-loading control. The inserted numbers are presented as relative expression folds in comparison to the cells without LLME treatment group after normalization to GAPDH. (F) The CSC activity of H1299 cells under JPH203 treatment with or without the overexpression of the myr-Akt vector was determined by tumorsphere cultivation, and pictures were presented at 100× magnification. The inserted scale bars indicate 100 µm in length. The quantitative data were presented as relative percentages of the tumorsphere number of the control vector transfected and 0.1% DMSO treatment group. The overexpression of myr-Akt (upper panel) was determined by Western blot analysis. ** p < 0.01. ns, not significant.

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Inhibition, Activity Assay, Activation Assay, Western Blot, Control, Expressing, Comparison, Over Expression, Plasmid Preparation, Transfection

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 4. LAT1 activity participates in PD-L1 expression on NSCLC cells. (A,B). The knockdown of LAT1 by lentiviral delivery of LAT1-specific shRNAs (shSLC7A5#1 or shSLC7A5#2) for A400 cells or by transfection of LAT1-specific siRNA oligonucleotides (si-SLC7A5) for H1299 cells was confirmed by Western blot analysis (A). The cell surface expressions of PD-L1 were determined by Western blot (A) or FACS analysis (B), respectively. The FACS data was analyzed by FlowJo software. The inserted numbers in (A) represent the relative expression levels of PD-L1 or LAT1 in comparison to the sh-LacZ or si-NC (indicated as 0 nM) control group, respectively, after normalization with GAPDH. Blue peaks and red peaks in (B) represent an isotype control and an anti-PD-L1 antibody, respectively. siNC, negative control siRNA. (C,D) The PD-L1 total protein and cell surface expressions of A400 and H1299 cells under JPH203 treatment were determined by Western blot (C) and FACS analysis (D), respectively. The inserted numbers in (A) present the relative expression levels of PD-L1 or LAT1 in comparison to the 0.1% DMSO control group (0 µM) after normalization with GAPDH. Blue peaks and red peaks in (D) represent an isotype control and an anti-PD-L1 antibody, respectively.

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Activity Assay, Expressing, Knockdown, Transfection, Western Blot, Software, Comparison, Control, Negative Control

Journal: International journal of molecular sciences

Article Title: L-Type Amino Acid Transporter 1 Regulates Cancer Stemness and the Expression of Programmed Cell Death 1 Ligand 1 in Lung Cancer Cells.

doi: 10.3390/ijms222010955

Figure Lengend Snippet: Figure 6. LAT1+/PD-L1+ enriches CSC activity of NSCLC cells. (A) The cell surface expressions of LAT1 and PD-L1 on A549 and H1299 cells were determined by FACS with fluorescence-labeled antibodies. (B) Three populations of NSCLC cells (PD-L1−/LAT1−, PD-L1+/LAT1−, PD-L1+/LAT1+) were isolated by FACS cell sorting, and their CSC activities were measured by tumorsphere cultivation. Pictures were presented at 100× magnification and the inserted scale bars indicate 100 µm in length. The data were presented as relative percentages of the tumorsphere number of PD-L1−/LAT1−cells. * p < 0.05. (C) The expressions of cancer stemness proteins (BMI1, c-MYC, EZH2, OCT4), phosphor-mTORser2481, mTOR, phosphor-Aktser473, and Akt were determined by Western blot. The inserted numbers indicated relative expression levels in comparison to PD-L1−/LAT1−cells after normalization to GAPDH. (D) The overall survival curves of NSCLC patients in the TCGA database regarding expression levels of CD274 and the two-gene signature of CD274 and SLC7A5 using median level as the cutoff criteria were analyzed using the GEPIA2 webtool [18].

Article Snippet: The PerCP conjugated mouse monoclonal anti-LAT1 antibody (clone name: BU53) was purchased from Novus Biologicals, LLC. (Centennial, CO, USA).

Techniques: Activity Assay, Labeling, Isolation, FACS, Western Blot, Expressing, Comparison