Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: Circulating Gal3 is a potential target for the diagnosis of LNM. TCGA database analysis of the expression level of Gal3 in serval solid cancer indicated that Gal3 is 100% positive in thyroid cancer, which is more than the rate in other cancers (A). The protein level of Gal3 in the serum of PTC patients was detected by ELISA analysis, showing that LNM patients express more Gal3 (B). Gal3 expression is significantly related to LNM based on univariant analysis in 50 PTC patients (C). Sequencing TCGA data downloaded from the LinkedOmics website demonstrated that the RNA level of Gal3 is upregulated in tumor tissues (D), and is associated with the stage of PTC (E) and LNM (F). Unsupervised hierarchical clustering heat map shows the expression of different genes between non-LNM and LNM PTC samples (G). Unpaired t-test and One-way ANOVA multiple comparisons test were performed to obtain P values in comparisons of two or more groups. * indicates P<0.05.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: Relationship between Gal3 expression and pathological features in PTC patients with or without LNM

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: Enrichment of miRNA and circRNA sequencing profiles. Flow chart of the circRNA/miRNA/mRNA screen to identify regulators upstream of Gal3 (A). Pathological morphology was validated by HE staining of two paired primary and LNM tumor samples that were prepared for RNA sequencing (B). Unsupervised hierarchical clustering shows distinct miRNA (C) and circRNA (D) expression with green representing decreased expression. Significantly enriched pathways in genes regulated by differentially expressed cancer-related miRNAs (E) and circRNAs (F) were identified by KEGG pathway enrichment analysis. Western blot analysis for ErbB, NOTCH, NF-κB and TGF-β pathways (G). IF staining for Gal3 and ErbB4 in original tumor and Lymph node metastasis tissues (H). bar = 20 μm.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Sequencing, Staining, RNA Sequencing, Expressing, Western Blot

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: miR-873 was identified to act as an upstream regulator of Gal3. Unsupervised hierarchical clustering heat map of miRNA expression fold-change from TCGA data shows the top 50 miRNAs with decreased expression in the LNM group. Nine miRNAs were consistent with our sequencing data, as shown by the black arrow (A). A Venn diagram showing that miR-873 is a potential regulator of Gal3 and is related to LNM in PTC (B). The putative binding site of miR-873-5p on Gal3 is shown, including the wild-type and mutated Gal3 3’-UTR sequences which were used to create the wild-type and mutant luciferase reporter constructs. The similarity between 12 putative complementary binding sites (red letters) of the Gal3 3’-UTRs from different species (C). A luciferase reporter assay demonstrated that miR-873 inhibited the transcription of the wild-type but not the mutant 3’-UTRs of Gal3 (D). The expression of endogenous Gal3 RNA was inhibited in miR-873-mimic-infected HEK293 cells compared to the control, as detected by qRT-PCR and normalized to GAPDH mRNA (E), while it was increased in the miR-873 inhibitor group (F). The RNA level of miR-873 was validated in LNM-positive tumor tissues compared to the negative control by analyzing the TCGA profile. N0: non-LNM; N1: LNM (G). miR-873 was validated again in a different metastatic lymph node group, N0: non-LNM; N1<4; 410 (H). Relative expression of miR-873 in circulation was detected by qRT-PCR between non-LNM and LNM patients (I). Unpaired t-test and one-way ANOVA multiple comparisons test were performed to obtain P values in comparisons of two or more groups. * indicates P<0.05.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Luciferase, Construct, Reporter Assay, Infection, Control, Quantitative RT-PCR, Negative Control

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: CircRNA UMAD1 is associated with LNM and Gal3 expression. Expression of hsa-circ-UMAD1 quantified by at least three independent qRT-PCR experiments in the cohort comparing non-LNM tumors (absent, n = 23) and LNM tumors (present, n = 27). Unpaired t-tests were performed to identify significant differences. The relative expression level of circRNA UMAD1 was normalized to the level of β-actin (A). CircRNA UMAD1 expression was significantly related to LNM and primary side location based on univariant analysis of 50 PTC patients, which is described in Table 1 (B). The correlation between the expression of circRNA UMAD1 and Gal3 was evaluated by Person’s correlation test (r = 0.35, P = 0.00138) (C). All data are expressed as the median ± range with three independent experiments.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Expressing, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: ROC curves for individual biomarkers circRNA UMAD1 and Gal3 or combination. ROC curve analysis of circRNA UMAD1 calculates the best cutoff point to discriminate between the LNM and primary tumor groups. The area under the curve (AUC) (SE) = 0.718 (0.072); 95% confidence interval was 0.576 to 0.86; P = 0.0084 (A). ROC curve analysis of Gal3 between these two groups. AUC (SE) = 0.825 (0.061); 95% confidence interval was 0.704 to 0.945; P<0.001 (B). ROC curve analysis of circRNA UMAD1 combined with Gal3 for distinguishing patients in the LNM vs. primary tumor groups. AUC (SE) = 0.87 (0.051); 95% confidence interval was 0.766 to 0.97; P<0.001 (C). Likelihood ratio = 3.75. P<0.01, ROC, receiver operating characteristic; SE, standard error.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques:

Journal: Cell Death & Disease

Article Title: Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics

doi: 10.1038/cddis.2016.239

Figure Lengend Snippet: Surface Gal3 defines a subtype of epithelial CSC. ( a ) Colorectal cancer cells LiM6 and DLD1 were investigated by flow cytometry for cell surface markers CD24/CD44 (left). CD24+/CD44+-cells (in black) were then investigated for CD166/EpCAM-expression (middle) and CD24+/CD44+/CD166+/EpCAM+-positive cells (in black middle) for Gal3-expression (right panel), where ++++ Gal3 − in green refers to cells positive for CD24, CD44, CD166, and EpCAM, but negative for Gal3, and ++++ Gal3 + in red refers to cells positive for CD24, CD44, CD166, EpCAM, and Gal3. ( b ) Pancreatic cancer cell lines AsPC1 and L3.6pl were subjected to the same analysis as in ( a )

Article Snippet: Proteins were detected using rat monoclonal antibody against Gal3 (1:500, ascites fluid of TIB-166, ATCC# M3/38.1.2.8 HL.2) and beta-actin (1:5000 AC-15, Sigma,A5316).

Techniques: Flow Cytometry, Expressing

Journal: Cell Death & Disease

Article Title: Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics

doi: 10.1038/cddis.2016.239

Figure Lengend Snippet: Gal3 positive CSC subset displays increased stemness characteristics. ( a ) ALDH activity was evaluated by flow cytometric analysis in Gal3 positive CSC (in red) and Gal3 negative CSC (in green). ALDH negative fractions are illustrated in gray. ( b ) Sphere formation ability (SFA) was determined. Gal3 positive CSC (red) accounted for significantly more spheres than Gal3 negative CSC (green). ( c ) TRAIL-resistance in Gal3 positive /Gal3 negative colon cancer CSC subsets was determined by APO-BRDU assay. Apoptotic cells are displayed as percentage of total cell count. ( d ) TRAIL-resistance in Gal3 positive /Gal3 negative CSC pancreatic cancer subsets. All experiments were performed in triplicate (see text for details). P <0.05 (*), P <0.01 (**), and P <0.001 (***)

Article Snippet: Proteins were detected using rat monoclonal antibody against Gal3 (1:500, ascites fluid of TIB-166, ATCC# M3/38.1.2.8 HL.2) and beta-actin (1:5000 AC-15, Sigma,A5316).

Techniques: Activity Assay, BrdU Staining, Cell Counting

Journal: Cell Death & Disease

Article Title: Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics

doi: 10.1038/cddis.2016.239

Figure Lengend Snippet: Elevated levels of Gal3 correlate with higher number of CSC marker positive cells. ( a ) Left: Venn diagrams, where areas represent percentage of total cell population, display surface Gal3-positive (blue plus red), CSC (green plus red), and their intersection (red) in LiM6 or LiM6TR. Right: SFA was determined in parental LiM6 and LiM6TR cells. White bars, spheres of 200–300 μ m; black bars, spheres of >300 μ m. ( b ) Left: Venn diagrams of expression of Gal3 and CSC markers in DLD1 and DLD1TR. Right: SFA was determined in parental DLD1 and DLD1TR cells. White bars, spheres of 75-150 μ m; gray bars, spheres of >150 μ m. ( c ) Lineage-tracking experiment outline: Gal3 positive CSC was transduced with an mCherry-lentiviral vector, Gal3 negative CSC with an eGFP-lentiviral vector and single cells of both mixed at different ratios. ( d ) SFA of these ratios was determined. Representative pictures of 100% Gal3 positive CSC (left), 100% Gal3 negative CSC (right), or 1:1 mixture (middle) are shown. P <0.05 (*); P <0.01 (**) and P <0.001 (***)

Article Snippet: Proteins were detected using rat monoclonal antibody against Gal3 (1:500, ascites fluid of TIB-166, ATCC# M3/38.1.2.8 HL.2) and beta-actin (1:5000 AC-15, Sigma,A5316).

Techniques: Marker, Expressing, Transduction, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics

doi: 10.1038/cddis.2016.239

Figure Lengend Snippet: Silencing of Gal3 shifts CSC subset to a Gal3 negative CSC subset. ( a ) Western blot analysis for comparison of total Gal3 in LiM6TR or DLD1TR colon cancer cells and L3.6pl or AsPC1 pancreatic cancer cells after infection with lentiviral particles for control-shRNA ( c ) or Gal3-shRNA (G). Each pair of control and Gal3 knockdown cells were run side by side on the same gel. Comparison of Gal3 expression between cell lines has been previously published ( b ) Flow cytometric analysis of colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (red trace) or Gal3-shRNA (green trace). Black trace is background staining. ( c ) ALDH activity was analyzed by flow cytometry. ALDEFLUOR activity in colon cancer and pancreatic cancer cells after infection with lentiviral particles for control-shRNA (blue trace) or Gal3-shRNA (green trace). Gray profile is background staining

Article Snippet: Proteins were detected using rat monoclonal antibody against Gal3 (1:500, ascites fluid of TIB-166, ATCC# M3/38.1.2.8 HL.2) and beta-actin (1:5000 AC-15, Sigma,A5316).

Techniques: Western Blot, Comparison, Infection, Control, shRNA, Knockdown, Expressing, Staining, Activity Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics

doi: 10.1038/cddis.2016.239

Figure Lengend Snippet: Effect of silencing of Gal3 on stemness behavior. ( a ) Knock-out of Gal3 (G-SH, in green) resulted in markedly decreased SFA compared to control cells (C-SH) in all cell lines. ( b ) Drug-resistance was significantly reduced in Gal3-depleted cells (G-SH, in green) as evaluated by APO-BRDU assays with conventional chemotherapy regimens (FOLFOX and FOLFIRI in colorectal cancer, left panels; gemcitabine in pancreatic cancer, right panel). ( c ) Tumor growth of Gal3 positive CSC (red) and Gal3 negative CSC LiM6TR cells (green) in subcutaneous model is shown. Gal3 positive CSC grow more rapidly with inoculation of 1 × 10 4 (upper curve) and 1 × 10 3 (lower curve) cells. ( d ) Representative picture of mice inoculated with 1 × 10 4 cells derived from LiM6TR. Gal3 positive (red arrow), Gal3 negative (green arrow). All experiments were performed in triplicate. P <0.05 (*), P <0.01 (**), and P <0.001 (***)

Article Snippet: Proteins were detected using rat monoclonal antibody against Gal3 (1:500, ascites fluid of TIB-166, ATCC# M3/38.1.2.8 HL.2) and beta-actin (1:5000 AC-15, Sigma,A5316).

Techniques: Knock-Out, Control, Derivative Assay