Journal: Journal of proteome research

Article Title: Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

doi: 10.1021/pr300483r

Figure Lengend Snippet: Immunocytochemical staining of TMPRSS4. In the top left panel, the domain structure of TMPRSS4 is shown, where TM refers to the transmembrane domain. The left bottom panel shows a CMA slide stained with a monoclonal antibody directed against TMPRSS4. The panel on the right shows a magnified view of four cell lines showing different levels of expression of TMPRSS4.

Article Snippet: CMA cell microarray Ep-CAM epithelial cell adhesion molecule CA19-9 carbohydrate antigen 19-9 HLA human leukocyte antigen TMPRSS4 transmembrane protease, serine 4 IHC immunohistochemistry ICC immunocytochemistry TMA tissue microarray ATCC American Type Culture Collection

Techniques: Staining, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Transcriptomic analysis identifies ST6GAL1 and ST3GAL3 as enriched in the cancerous ducts. A , biosynthetic pathways for glycans underlying select lectin signatures are shown. ST3GALs are responsible for transferring α-2,3-sialosides and ST6GAL1/2 for α-2,6-sialosides, and MGAT3 for bisecting GlcNAc. The glycans are annotated following the Symbolic Nomenclature for Glycans. B , transcriptomic analysis assessing the mRNA levels of select glycosyltransferases between normal adjacent and matched cancerous tissues within the same patient. C , uMAP plots representing the cells isolated from patients with PDAC (n = 24) and normal pancreata (n = 11) pooled on single-cell sequencing. The clusters representing the normal ductal cells and tumor ductal cells are highlighted. D , violin plot showing the comparison of ST6Gal1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. The violin plots showing the comparison of ST6Gal1 and ST3GAL1 levels in normal ( green ) versus PDAC ( blue ) ductal clusters. The clusters in each group are combined. ns, p > 0.05; ∗ p < 0.05; student’s t test ( two-tailed ). MGAT3, beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; ST3GAL3, ST3 beta-galactoside alpha-2,3-sialyltransferase 3; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; uMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Transferring, Isolation, Sequencing, Comparison, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Profiling of ST6GAL1 and SNA in human pancreatic cancer shows association with the stage and survival. A , H&E of the normal pancreas ( left ), stage I pancreatic adenocarcinoma ( center ), and stage IV PDAC ( right ) stained from a BioMax human tissue microarray. B , multiplex OPAL IF staining of SNA ( yellow ), ST6GAL1 ( red ), and DAPI ( blue ) on the corresponding normal pancreas and stage I and stage IV pancreatic adenocarcinoma purchased from BioMax human tissue microarray. The scale bars represent 25 μm. C , quantification of SNA-positive cells per high-powered field based on multiplex IF in normal versus all cancerous cases in human tissue microarray. Owing to the high number of positive cells, each HPF was given a score (1–3) based on the number of SNA-positive cells per field. D , quantification of ST6GAL1-positive cells per high-powered field in normal versus all cancerous cases in human tissue microarray. E , distribution of the total ST6GAL1-positive cells per high-powered field across normal and each stage of pancreatic cancer based on multiplex IF imaging of human pancreatic cancer tissue microarray. Statistical significance was evaluated using the Student’s t test ( two-tailed ): ns, p > 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. HFP, high-powered field; ns, not statistical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1.

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Staining, Microarray, Multiplex Assay, Imaging, Two Tailed Test

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Integrated Systems Analysis of the Murine and Human Pancreatic Cancer Glycomes Reveals a Tumor-Promoting Role for ST6GAL1

doi: 10.1016/j.mcpro.2021.100160

Figure Lengend Snippet: Pancreas-specific deletion of ST6GAL1 reduces disease burden in murine PDAC. A , breeding schematic illustrating the generation of novel ST6KC mice. The offspring of parental strains crossed into p48-Cre mice drive the induction of mutant KRAS G12D and deletion of ST6GAL1 under the same promoter. B , IHC staining for ST6GAL1 ( brown ) in KC and ST6KC mice (n = 3 per group). The number of ST6GAL1+ cells per high-powered field is quantified. The scale bar represents 50 μm. C , IF staining for SNA ( yellow ) and DAPI ( blue ) in KC and ST6KC mice (n = 3 per group). The number of SNA+ cells per high-powered field is quantified. The scale bar represents 100 μm. D , H&E of 14-week-old FFPE pancreata from KC and ST6KC mice (n = 5 per group). The percent of the preserved normal pancreas area is quantified per high-powered field. The scale bar represents 200 μm. E , trichrome and gomori ( blue ) stain of FFPE pancreata from 14-week-old KC and ST6KC mice (n = 5 per group). The percent of collagen deposition fibrosis is quantified per high-powered field on the right. The scale bar represents 200 μm. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student’s t test ( two-tailed ). FFPE, formalin-fixed paraffin-embedded; IF, immunofluorescence; IHC, immunohistochemical; PDAC, pancreatic ductal adenocarcinoma; SNA, Sambucus nigra agglutinin; ST6GAL1, ST6 beta-galactoside alpha-2,6-sialyltransferase 1; ST6KC, ST6GAL1 flx/flx ;p48 Cre ; LSL KRASG12D .

Article Snippet: The slide was then incubated with anti-human ST6GAL1 (Proteintech, cat # 14355-1-AP) followed by Opal Polymer horseradish peroxidase Ms + Rb (Akoya Biosciences, cat # ARH1001EA) and tyramide-linked 650 Opal fluorophore (Akoya Biosciences, cat # FP1496001KT) to amplify signals.

Techniques: Mutagenesis, Immunohistochemistry, Staining, Two Tailed Test, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Immunohistochemical staining

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 2 Validation of clusterin upregulation by semi-quantitative reverse transcription–PCR, western blot analysis and immunofluorescence microscopy. (a) Semi-quantitative reverse transcription–PCR analysis (bottom panels) of the clusterin transcript (CLU) in BRI-JM01 cells induced after 2, 4, 6 and 24 h of TGF-b1 (100 pM) exposure confirming the clusterin transcript modulation observed in the microarray experiments (The graph underneath represents normalized data obtained from the microarray experiments. Bars represent the t-test P-value of 4–6 experiments performed for each time point.) The Eef1a-1 transcript (top panel), which does not change upon TGF-b1 treatment, was used as loading control. (b) Western blot analysis of whole-cell lysates (WCL) from BRI-JM01 cells grown in the absence or presence of 100 pM TGF-b1 (24 h) confirming the upregulation of clusterin and showing the uncleaved precursor form (pCLU) of clusterin in the WCL, and the the mature processed form (sCLU) in both the WCL and the CM. Membranes containing the WCL samples were reprobed for b-actin to confirm equal loading. (c) Immunofluorescence microscopy (magnification 1000) of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h) show clusterin to be close to the cell’s outer membrane (top panels) and (d) colocalized with the Golgi marker b-COP (bottom) (red, clusterin; green, b-COP; blue, diamidino phenylindole (DAPI)-stained nuclei; magnification 400). (e) Western blot of the CM confirming the increased levels of secreted clusterin (sCLU) in the medium of BRI-JM01 cells treated with 100 pM TGF-b1 (24 h). CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Biomarker Discovery, Reverse Transcription, Western Blot, Microscopy, Microarray, Control, Membrane, Marker, Staining

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 3 Antibodies against sCLU block the TGF-b1-induced loss of junctional ZO-1 and E-cadherin in BRI-JM01 cells. Immunofluorescence microscopy of ZO-1 in BRI-JM01 cells grown (a) in the absence or presence of 100 pM TGF-b1 with or without antibodies against TGF-b (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml), or (b) in CM from untreated BRI-JM01 cells (CM-CTL) or from cells treated with 100 pM TGF-b1 for 24 h (CM-TGF-b1) both in the absence or presence of antibodies against (anti-TGF-b, 10 nM) or clusterin (anti-clu, 8 mg/ml). For all the panels: red, ZO-1; blue, diamidino phenylindole (DAPI)-stained nuclei (magnification: 400). (c) Flow cytometric evaluation of the cell-surface levels of E-cadherin expressed by BRI-JM01 cells exposed to TGF-b1 (100 pM) in the absence or presence of clusterin polyclonal IgG (anti-clu, 8 mg/ml). Cell populations expressing high ( þ ) and low () levels of cell-surface E-cadherin are indicated. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Blocking Assay, Microscopy, Staining, Expressing

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 4 Purified clusterin promotes a spindle-shaped morphol- ogy and loss of junctional ZO-1 in BRI-JM01 cells. (a) Coomassie blue-stained gel under non-reducing (DTT (dithiothreitol)) and reducing ( þ DTT) conditions of purified recombinant human clusterin (b) expressed and secreted by HEK-293-E6 cells. (b) BRI- JM01 cells treated with 200 nM purified clusterin (24 h) show a spindle-shaped morphology (top; magnification 40) and loss of tight-junctional ZO-1 (red, bottom; magnification 400). HEK, human embryonic kidney; ZO, zona occludens.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Staining, Recombinant

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 6 Clusterin polyclonal IgG inhibits the invasive behavior of cell lines other than the BRI-JM01 cell line. Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits (a) the stellate morphology of 4T1 and PC3 tumor cells when cultured in Matrigel for 3 weeks (magnification 40). (b) Western blot analysis confirming the presence of sCLU in the CM of 4T1, NMuMG and PC3 cells. (c) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of 4T1, NMuMG and PC3 cell lines ing a Transwell invasion assay. Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. (d) Clusterin polyclonal IgG (anti-clu, 8 mg/ml) does not significantly affect the growth-inhibitory response induced by TGF-b1 in 4T1, NMuMG and PC3 cells. Results are expressed relative to non-treated cells and are shown as the average (±s.d.) of two independent experiments performed in triplicate. (e) Western blot analysis showing the presence of sCLU in the CM of MDA-MB231LM2 cells (left panel). Clusterin polyclonal IgG (anti-clu, 8 mg/ml) inhibits the Matrigel invasion of these cells in a Transwell assay (right panel). Results (expressed relative to non-treated cells) are shown as the average (±s.d.) of two independent experiments. CM, conditioned medium; sCLU, secreted clusterin; TGF-b, transforming growth factor-b.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Cell Culture, Western Blot, Transwell Invasion Assay, Transwell Assay

Journal: Oncogene

Article Title: Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

doi: 10.1038/onc.2009.399

Figure Lengend Snippet: Figure 7 Clusterin monoclonal antibodies inhibit the motility of 4T1 cells and significantly reduce 4T1 cell metastasis to lungs after orthotopic implantation. (a) Evaluation of the ability of in-house-generated clusterin monoclonal antibodies 11E2, 16B5, 16C11 and 20G3 at a concentration of 8 mg/ml (black bars), and a commercially available clusterin monoclonal antibody B5 and anti-peptide polyclonal IgG, C18 (white bars), to block 4T1 cell motility in a BCSM assay. The average motility (±s.d.) was determined by measuring ink clearance in 10 independent microscopic fields (per treatment) and is expressed as average ink clearance/cell/24 h relative to non-treated control cells (gray bar). The hatched line depicts the cut-off that we used to define an antibody as having neutralizing activity, which was based on the degree of inhibition caused by B5 antibody. (b) Experimental design. 4T1 cells (4 104) were injected in the left # 4 inguinal mammary gland. Animals were treated (5 mg/kg) with neutralizing (16B5, 16C11 and 11E2) clusterin antibody, non-neutralizing (20G3) clusterin antibody or saline (control) thrice a week (intraperitoneally), starting the day of cell implantation. Mice were killed on day 28. (c) The number of macroscopically grossly visible lung metastasis in the mice were quantified 28 days post tumor cell implantation and statistically analysed using the non-parametric Mann–Whitney U-test (n ¼ 10 for the saline, 20G3 and 11E2 groups; n ¼ 9 for the 16B5 and 16C11 groups). BCSM, black cellular spreading and motility.

Article Snippet: Antibodies against the following proteins were purchased: clusterin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), E-cadherin (Sigma, Minneapolis, MN, USA; R&D Systems, Oakville, Canada), ZO-1 (anti-ZO1; Chemicon, Billerica, MA, USA; Zymed, San Francisco, CA, USA), b-COP (anti-b-COP; Cedarlane, Burlington, Canada) smad2 and phospho-smad2 (both Cell Signaling Technology, Danvers, MA, USA) and b-actin (Sigma).

Techniques: Bioprocessing, Generated, Concentration Assay, Blocking Assay, Control, Activity Assay, Inhibition, Injection, Saline, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Mesenchymal stromal cell-derived extracellular vesicles reduce lung inflammation and damage in nonclinical acute lung injury: Implications for COVID-19

doi: 10.1371/journal.pone.0259732

Figure Lengend Snippet: (A) Antibody microarray particle analysis of tetraspanin proteins. (B) Electrochemiluminescent multiplexed immunoassay detection of tetraspanin proteins. (C) Western blot analysis of MSC-EV proteins flotillin-1, Annexin-2, Syntenin-1, MHC-I and contaminating proteins MHC-II and calreticulin. (D) Immunogold cryo-transmission electron microscopy imaging for CD63 and phosphatidylserine. Atomic force microscopy for MSC-EV morphology and size. (E) Nanoparticle tracking for MSC-EV size and concentration. (F) MSC-EV particle density correlated to phospholipid content. Error bars are SEM of the mean. *p ≤0.05 vs CD63. Scale bar denotes 100 nm.

Article Snippet: Lysates were volume loaded and separated on a 12% Bolt Bis-Tris Gel and probed using Abcam antibodies against flotillin-1 (ab133497 at 1:10,000 dilution), Annexin-2 (ab41803 at 1:1000 dilution), Syntenin-1 (ab19903 at 1:1000 dilution), MHC-I (ab110645 at 1:1000 dilution), MHC-II (157210 at 1:10000 dilution), and Calreticulin (ab92516 at 1:1000 dilution).

Techniques: Microarray, Western Blot, Transmission Assay, Electron Microscopy, Imaging, Microscopy, Concentration Assay

Journal: Sensors & Diagnostics

Article Title: A novel point-of-care diagnostic prototype system for the simultaneous electrochemiluminescent sensing of multiple traumatic brain injury biomarkers †

doi: 10.1039/d3sd00090g

Figure Lengend Snippet: Microarray-type SR-ECLIA 3-plex protocol: a) schematic representation of the assay established for detection of three mTBI biomarkers (GFAP, h-FABP, S100β) on a single SPCE. STEP 1 includes the spotting of small quantities of capture antibodies of each biomarker on the predefined spot positions on the SPCE using a spotter device (BSA labelled with SULFO-TAG was used for control/alignment purposes); STEP 2 includes the typical sandwich assay protocol composed of a blocking step, addition of three antigens, and addition of detection antibodies modified with the SULFO-TAG label carrying Ru(bpy) 3 2+ ; STEP 3 is the detection step that includes addition of the tripropylamine (TPA) co-reactant and applying a potential on the SPCE to trigger the ECL signal generation from each biomarker spot; STEP 4 comprises the raw data processing; b) optimization of capture antibody amount per spot (cAb amount for all biomarkers: 7.00 ng per spot, 2.10 ng per spot, 0.70 ng per spot, 0.18 ng per spot). The antigen concentrations were 0, 1, 10 and 50 ng mL −1 . The other assay conditions are listed in SI-5.

Article Snippet: Antigen S100BB homodimer and S100A1B heterodimer human (ref. 8S9h, HyTest Ltd., Turku, Finland); monoclonal mouse anti-human S100 proteins (ref. 4S37, HyTest Ltd., Turku, Finland) clone 8B10cc and clone 6G1cc were employed as capture and detection antibody, respectively.

Techniques: Microarray, Biomarker Discovery, Control, Blocking Assay, Modification

Journal: Sensors & Diagnostics

Article Title: A novel point-of-care diagnostic prototype system for the simultaneous electrochemiluminescent sensing of multiple traumatic brain injury biomarkers †

doi: 10.1039/d3sd00090g

Figure Lengend Snippet: Microarray-type SR-ECLIA 3-plex assay performance in 50% serum (v/v) using POC prototype detection system: a) capture antibody spotting pattern (letter “C” – BSA@SULFO-TAG control spots; letter “G” – GFAP biomarker spots; letter “H” – h-FABP biomarker spots; letter “S” – S100β biomarker spots); and corresponding ECL raw data images generated at various biomarker concentrations (0, 250, 500, 750, and 1000 pg mL −1 ) (8 × 8 binning and dark subtraction, but no other postprocessing), and b) obtained 3-plex calibration curves. The error bars represent the standard deviations from two replicates ( n = 2) and the fitting was performed with a 4-PL equation; c) assay specificity heatmap at single antigen concentrations of 2.5 ng mL −1 .

Article Snippet: Antigen S100BB homodimer and S100A1B heterodimer human (ref. 8S9h, HyTest Ltd., Turku, Finland); monoclonal mouse anti-human S100 proteins (ref. 4S37, HyTest Ltd., Turku, Finland) clone 8B10cc and clone 6G1cc were employed as capture and detection antibody, respectively.

Techniques: Microarray, Plex Assay, Control, Biomarker Discovery, Generated

Journal: Sensors & Diagnostics

Article Title: A novel point-of-care diagnostic prototype system for the simultaneous electrochemiluminescent sensing of multiple traumatic brain injury biomarkers †

doi: 10.1039/d3sd00090g

Figure Lengend Snippet: Results of the inter-assay variability (CV) for the 3-plex SR-ECLIA for three independent experiments made in duplicate measurements. Preliminary recovery results for the mTBI biomarkers with 50% human serum (v/v) in assay diluent spiked at 800 pg mL −1 . The calculations were made with calibration curves generated in complex matrix

Article Snippet: Antigen S100BB homodimer and S100A1B heterodimer human (ref. 8S9h, HyTest Ltd., Turku, Finland); monoclonal mouse anti-human S100 proteins (ref. 4S37, HyTest Ltd., Turku, Finland) clone 8B10cc and clone 6G1cc were employed as capture and detection antibody, respectively.

Techniques: Inter Assay, Generated, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Capability of Human Dendritic Cells Pulsed with Autologous Induced Pluripotent Stem Cell Lysate to Induce Cytotoxic T Lymphocytes against HLA-A33-Matched Cancer Cells

doi: 10.3390/ijms232112992

Figure Lengend Snippet: Heatmap analysis of gene expression of iPSCs, SW48 and T47D cells, and fibroblasts. The iPSC data were obtained from microarray analysis. The SW48 and T47D cell and fibroblast data were obtained from the NCBI GEO database. The listed genes are highly expressed in iPSCs and are very lowly or not expressed in fibroblasts (gene expression level < 5). The change from red to light purple indicates the gene expression level changing from 15 to 0. The 99 genes in the list were selected in order of highest gene expression in the iPSCs.

Article Snippet: The SW48 (colorectal cancer: HLA-A33), T47D (breast cancer: HLA-A33), and T98G (glioblastoma: HLA-A02) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Gene Expression, Microarray

Journal: International Journal of Molecular Sciences

Article Title: Capability of Human Dendritic Cells Pulsed with Autologous Induced Pluripotent Stem Cell Lysate to Induce Cytotoxic T Lymphocytes against HLA-A33-Matched Cancer Cells

doi: 10.3390/ijms232112992

Figure Lengend Snippet: TAAs selected by the NCI Pilot Project. The list shows the ranking of TAAs selected as described previously . The iPSC data were obtained using microarray analysis. The iPSC, SW48 and T47D cell, and fibroblast data were obtained from the NCBI GEO database. The change from red to light purple indicates the gene expression level changing from 15 to 0.

Article Snippet: The SW48 (colorectal cancer: HLA-A33), T47D (breast cancer: HLA-A33), and T98G (glioblastoma: HLA-A02) cell lines were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Microarray, Gene Expression

Journal: Carcinogenesis

Article Title: Paradoxic effects of metformin on endothelial cells and angiogenesis

doi: 10.1093/carcin/bgu001

Figure Lengend Snippet: Metformin modulates several angiogenesis-related genes. Microarray analysis was performed on total RNAs isolated from HUVE cells from three different donors that were treated with either vehicle alone (CTR) or 6 or 24h with 10mM metformin (Metf). Data from the scans were normalized and analyzed for relative intensity of regulation of genes with metformin treatment. ( A ) Hierarchical clustering of human endothelial cells from the three different donors shows clear division into control, 6 and 24h metformin treatment, genes showing statistically significant regulation in all donors are shown. The genes are as follows: VEGF-A ; PTGS2 (COX2); F3 (coagulation factor III, thromboplastin); ERG (ETS-related gene); FLT1 (VEGF receptor 1); SAT1 (spermidine/spermine N1-acetyltransferase 1); PRKD1 (protein kinase D1) CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1); WARS (tryptophanyl-tRNA synthetase); ADAMTS1 ; CXCR4 ; and CCL2 (CC chemokine ligand 2). ( B ) Real-time PCR validation of gene expression of the statistically significant angiogenesis-related genes up or downregulated by metformin in microarrays. Relative expression values are indicated as fold change over untreated HUVE cells (=1) assessed after normalization on glyceraldehyde 3-phosphate dehydrogenase , RNA polymerase II and glucose-6-phosphate dehydrogenase expression data obtained from reactions run in parallel. All amplifications were performed in triplicate. ( C ) Analysis of CXCR4, CYP1B1, COX2, VEGF-A and ADAMTS1 protein expression on HUVE cells, by western blotting. A table summarizing protein changes relative to control (Ctrl) is also shown (upregulated proteins are shown in red; downregulated proteins are shown in green). β-Actin was used as loading control.

Article Snippet: Following blocking with 5% non-fat milk powder (wt/vol) in Tris-buffered saline (10mM Tris–HCl, pH 7.5, 100mM NaCl, 0.1% Tween-20) for 1h at room temperature, membranes were incubated with primary antibodies directed against the following human antigens: AMPKα1, phospho-AMPKα (Thr172), β-actin, total and phospho-NF-κB, cyclin D1, glyceraldehyde-3-phosphate dehydrogenase, vinculin, CDK4, phospho-extracellular signal-regulated kinase (ERK1/2), caspase 3 (all purchased from Cell Signaling Technology, Danvers, MA), CYP1B1 (Abcam, Cambridge, UK), CXCR4 (Santa Cruz Biotechnology, Santa Cruz, CA), ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS1; Millipore Corporation), cyclooxygenase 2 (COX2; Cayman Chemical, Ann Arbor, MI) and VEGF (Calbiochem).

Techniques: Microarray, Isolation, Control, Coagulation, Real-time Polymerase Chain Reaction, Biomarker Discovery, Gene Expression, Expressing, Western Blot

Journal: Carcinogenesis

Article Title: Paradoxic effects of metformin on endothelial cells and angiogenesis

doi: 10.1093/carcin/bgu001

Figure Lengend Snippet: Tumor cell supernatants upregulate CYP1B1 expression in HUVE cells, metformin abrogates this effect. HUVE cells were incubated with serum-free unconditioned medium (NC) or conditioned supernatants (conditioned medium, CM) from tumor cells (MDA-MB-231 for breast cancer; PC3 and DU-145 for prostate cancer), all harvested after 24h. ( A ) Western blotting shows a significant upregulation of CYP1B1 in HUVE cells cultured in presence of conditioned supernatants as compared with those treated with unconditioned medium. Addition of metformin significantly abrogates this effect. ( B ) HUVE cells were transfected with an AMPKα-specific or control (SiCTR) siRNAs, treated with or without 10mM metformin, lysed and analyzed by western blotting for CYP1B1 expression. CYP1B1 is significantly downregulated in AMPKα siRNA-transfected cells as compared with control siRNA. Metformin upregulates CYP1B1 levels in AMPKα siRNA-transfected HUVE cells, suggesting AMPK-dependent CYP1B1 expression in these cells. β-Actin was used as a loading control. The values in the western blot panels represented the ratio of the indicated protein and actin.

Article Snippet: Following blocking with 5% non-fat milk powder (wt/vol) in Tris-buffered saline (10mM Tris–HCl, pH 7.5, 100mM NaCl, 0.1% Tween-20) for 1h at room temperature, membranes were incubated with primary antibodies directed against the following human antigens: AMPKα1, phospho-AMPKα (Thr172), β-actin, total and phospho-NF-κB, cyclin D1, glyceraldehyde-3-phosphate dehydrogenase, vinculin, CDK4, phospho-extracellular signal-regulated kinase (ERK1/2), caspase 3 (all purchased from Cell Signaling Technology, Danvers, MA), CYP1B1 (Abcam, Cambridge, UK), CXCR4 (Santa Cruz Biotechnology, Santa Cruz, CA), ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS1; Millipore Corporation), cyclooxygenase 2 (COX2; Cayman Chemical, Ann Arbor, MI) and VEGF (Calbiochem).

Techniques: Expressing, Incubation, Western Blot, Cell Culture, Transfection, Control

Journal: Carcinogenesis

Article Title: Paradoxic effects of metformin on endothelial cells and angiogenesis

doi: 10.1093/carcin/bgu001

Figure Lengend Snippet: Metformin inhibits VEGF-induced ERK1/2 activation in an AMPK-dependent manner. HUVE cells were plated onto six-well plates, treated with or without VEGF in presence or absence of 10mM metformin and/or 10 µM compound C and analyzed at different time points by western blotting. ERK1/2 phosphorylation was measured at 5, 15, 30min or 6h after treatment. ( A ) ERK1/2 activation was reduced in metformin-treated cells, starting at 15min after metformin addition to the medium. ( B ) Metformin inhibited phosphorylation of ERK1/2 and inhibited VEGF-induced ERK1/2 phosphorylation. When compound C (cC) was added to the medium, the effects of metformin inhibition and induction of phosphorylation by VEGF alone were counteracted suggesting an involvement of AMPK in ERK1/2 signaling. The experiments were performed twice in triplicate with similar results. The values in the western blot panels represented the ratio of the indicated protein and actin; β-actin was used as loading control.

Article Snippet: Following blocking with 5% non-fat milk powder (wt/vol) in Tris-buffered saline (10mM Tris–HCl, pH 7.5, 100mM NaCl, 0.1% Tween-20) for 1h at room temperature, membranes were incubated with primary antibodies directed against the following human antigens: AMPKα1, phospho-AMPKα (Thr172), β-actin, total and phospho-NF-κB, cyclin D1, glyceraldehyde-3-phosphate dehydrogenase, vinculin, CDK4, phospho-extracellular signal-regulated kinase (ERK1/2), caspase 3 (all purchased from Cell Signaling Technology, Danvers, MA), CYP1B1 (Abcam, Cambridge, UK), CXCR4 (Santa Cruz Biotechnology, Santa Cruz, CA), ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS1; Millipore Corporation), cyclooxygenase 2 (COX2; Cayman Chemical, Ann Arbor, MI) and VEGF (Calbiochem).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Inhibition, Control