Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Galectin-3 functions as an adhesion molecule to support eosinophil rolling and adhesion under conditions of flow.

doi: 10.4049/jimmunol.179.11.7800

Figure Lengend Snippet: FIGURE 2. Eosinophil-expressed Gal-3 mediates eosinophil rolling and adhesion to VCAM-1. Eosinophils from allergic donors were preincubated with medium alone (n 16), mAbs against Gal-3 (n 3), 4 (n 10), or 2 (n 10), normal rat IgG (NrIgG; n 3), or normal mouse IgG (NmIgG; n 3) at 10 g/ml for 20 min and infused into the flow chamber containing rhVCAM-1-coated coverslips. The interactions of the injected cells with the VCAM-1-coated coverslips were recorded. In some experi- ments, eosinophils were preincubated with lactose (n 5) or maltose as a control (n 3) at 3 mM before infusion. Results shown represent com- bined data for each treatment expressed as mean SE of the number of rolling or adherent cells per hpf. , p 0.05.

Article Snippet: In some experiments, eosinophils were preincubated with mAbs against murine Gal-3 prepared from a rat hybridoma (M3/38.1.2.8 HL.2; catalog no. TIB-166 from American Type Culture Collection) that is known to specifically recognize human Gal-3 (33), anti-human 4 mAb P4G9 (Immunotech), anti-human 2 mAb 60.3 (34), normal rat IgG (Santa Cruz Biotechnology), normal mouse IgG (BD Pharmingen), or a combination of anti- 4 and anti-Gal-3 for 20 min before infusion into the flow chamber.

Techniques: Injection, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Galectin-3 functions as an adhesion molecule to support eosinophil rolling and adhesion under conditions of flow.

doi: 10.4049/jimmunol.179.11.7800

Figure Lengend Snippet: FIGURE 3. Eosinophil-expressed 4 integrins and Gal-3 mediate eosino- phil rolling and adhesion to Gal-3. Eosinophils from allergic donors were preincubated with medium alone (n 10), mAbs against Gal-3 (n 6), 4 (n 5) or 2 (n 5), normal rat IgG (NrIgG; n 3), or normal mouse IgG (NmIgG; n 3) before infusion into the flow chamber containing rhGal-3- coated coverslips, and the interactions of the injected cells with the coated coverslips were recorded. In some experiments, eosinophils were preincubated with lactose (n 9) or maltose as a control (n 4) before infusion. Results shown represent combined data for each treatment expressed as mean SE of the number of rolling or adherent cells per hpf. , p 0.05.

Article Snippet: In some experiments, eosinophils were preincubated with mAbs against murine Gal-3 prepared from a rat hybridoma (M3/38.1.2.8 HL.2; catalog no. TIB-166 from American Type Culture Collection) that is known to specifically recognize human Gal-3 (33), anti-human 4 mAb P4G9 (Immunotech), anti-human 2 mAb 60.3 (34), normal rat IgG (Santa Cruz Biotechnology), normal mouse IgG (BD Pharmingen), or a combination of anti- 4 and anti-Gal-3 for 20 min before infusion into the flow chamber.

Techniques: Injection, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Galectin-3 functions as an adhesion molecule to support eosinophil rolling and adhesion under conditions of flow.

doi: 10.4049/jimmunol.179.11.7800

Figure Lengend Snippet: FIGURE 5. Gal-3 expression on eosinophils from allergic donors is el- evated and is colocalized with 4 expression. A, Eosinophils (nonperme- abilized) from normal (n 3) and allergic donors (n 4) were adhered to VCAM-1-coated coverslips and incubated with FITC-conjugated anti- human Gal-3 or FITC-conjugated mouse IgG (control). The cells were fixed, mounted on glass slides, and observed by confocal microscopy. Original magnification was 20. B, Gal-3 expression on eosinophils from normal (n 3) and allergic (n 5) donors was analyzed by flow cytom- etry using mAb A3A12 against human Gal-3. The percentage of Gal-3 positive cells (mean SE) after subtracting the isotype is shown. , p 0.05. The inset shows a histogram of an overlay of Gal-3 expression by a representative allergic donor vs a normal donor. C, Eosinophils from al- lergic donors (n 3) were first exposed to anti-4 mAb P4G9 (10 g/ml)

Article Snippet: In some experiments, eosinophils were preincubated with mAbs against murine Gal-3 prepared from a rat hybridoma (M3/38.1.2.8 HL.2; catalog no. TIB-166 from American Type Culture Collection) that is known to specifically recognize human Gal-3 (33), anti-human 4 mAb P4G9 (Immunotech), anti-human 2 mAb 60.3 (34), normal rat IgG (Santa Cruz Biotechnology), normal mouse IgG (BD Pharmingen), or a combination of anti- 4 and anti-Gal-3 for 20 min before infusion into the flow chamber.

Techniques: Expressing, Incubation, Control, Confocal Microscopy

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: The regulation of p21 expression by Gal-3 in human prostate cancer cells. The expression levels of Gal-3 and p21 were analyzed by Western blot analysis. (a) Gal-3 over-expression in LNCaP cells up-regulated the endogenous level of p21 protein. (b) Gal-3 knockdown in DU145 cells down-regulated the endogenous level of p21. β-actin was used as the loading control. Data are representative of three independent experiments.

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Over Expression

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: p21 partially mediates the functions of Gal-3. The expression levels of active caspase-3, p21, Gal-3, and β-actin were analyzed by Western blot analysis. (a) Gal-3 over-expression-mediated inhibition of apoptosis was reversed by p21 knockdown. LNCaP VC and cl-2911 were transfected with p21 siRNA or control siRNA, 48 h later, cells were treated with cisplatin (50 μM) for 12 h. (b) Gal-3 knockdown-induced apoptosis was attenuated by p21 over-expression. DU145 VC and siGal3-35 were transfected with pcDNA 6.0-p21-V5His or control vector, 48 h later, cells were treated with cisplatin (50 μM) for 12 h. β-actin was used as the loading control. (c) Gal-3 knockdown led to increased cell growth of DU145. Cell growth was analyzed by MTT assay. **, P <0.01 vs. control vector. (d) Gal-3 knockdown-induced increase in cell growth of DU145 was slowed down by p21 over-expression. Cells were seeded into 96-well plate, the next day, transfected with pcDNA 6.0-p21-V5His or control vector, two days later, cell growth was analyzed by MTT assay. **, P <0.01 vs. siGal3-35 transfected with control vector. Data are representative of three independent experiments.

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Over Expression, Inhibition, Transfection, Plasmid Preparation, MTT Assay

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: Gal-3 enhances the stability of p21. (a) Gal-3 over-expression in LNCaP cells increased the stability of p21. (b) Gal-3 knockdown in DU145 cells decreased the stability of p21. Cells were treated with cycloheximide (50 μg/ml) for indicated times, collected and subjected to Western blot analysis. β-actin was used as the loading control. Data are representative of three independent experiments. The graph of half-life of p21 in LNCaP (c) and DU145 (d) cells. p21 expression was quantified by densitometric analysis and normalized to β-actin. The relative abundance of p21 was represented as the percentage remaining relative to time zero.

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Over Expression, Western Blot, Expressing

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: The stabilization of p21 by Gal-3 is impaired by GCS-100. LNCaP Cl-2911 (a) and DU145 parental cells (b) were treated with 1% GCS-100 or 1%CP for 24 h, and treated with cycloheximide (50 μg/ml) for indicated times. Cells were harvested and subjected to Western blot analysis. Data are representative of three independent experiments. The graph of half-life of p21 in LNCaP cl-2911 (c) and DU145 cells (d) after GCS-100 treatment. p21 expression was quantified by densitometric analysis and normalized to β-actin. The relative abundance of p21 was represented as the percentage remaining relative to time zero.

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: The stabilization of p21 by Gal-3 is impaired by lactose. LNCaP Cl-2911 (a) and DU145 parental cells (b) were treated with 100mM lactose or 100mM sucrose or remained untreated, 24 h later, treated with cycloheximide (50 μg/ml) for indicated times. Cells were harvested and subjected to Western blot analysis. β-actin was used as the loading control. Data are representative of three independent experiments. The graph of half-life of p21 in LNCaP cl-2911 (c) and DU145 (d) cells after lactose treatment. p21 expression was quantified by densitometric analysis and normalized to β-actin. The relative abundance of p21 was represented as the percentage remaining relative to time zero.

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: The effects of different Gal-3 fragments on p21 stability. Gal-3 null C4-2B cells were transfected with plasmids containing three different Gal-3 fragments sequence (1-107aa, 108-250aa, and 1-250aa). (a) Gal-3 (108-250) and Gal-3 (1-250) stabilized p21 protein in C4-2B cells. Cells were treated with cycloheximide (50 μg/ml) for indicated times, collected and subjected to Western blot analysis. Compared to control transfectants (I), p21 stability was increased in Gal-3 (108-250) (III) and Gal-3 (1-250) (IV) transfected clones, but not in Gal-3 (1-107) (II) transfected clone. β-actin was used as the loading control. Data are representative of three independent experiments. (b) The graph of half-life of p21 in Gal-3 fragments transfected clones. p21 expression was quantified by densitometric analysis and normalized to β-actin. The relative abundance of p21 was represented as the percentage remaining relative to time zero.

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Sequencing, Western Blot, Clone Assay, Expressing

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: The interaction of p21 protein with the CRD of Gal-3. (a) Detection of the interaction of p21 with the CRD of Gal-3 by Co-IP. LNCaP cl-2911 cells were treated with 100 mM lactose or remained untreated for 24 h, collected and analyzed by Co-IP as described in Materials and Methods. 1-4, immunoblot for p21; 1′-4′, immunoblot for Gal-3; 1 and 1′, untreated cell lysate IP with normal rat IgG; 2 and 2′, untreated cell lysate IP with rat anti-Gal-3; 3 and 3′, lactose treated cell lysate IP with rat anti-Gal-3; 4 and 4′, untreated total cell lysate. Prediction of the interaction of CRD of Gal-3 with p21 (b) and lactose (c) . The references about the structure of Gal-3 CRD, lactose, and p21 were indicated in Meterials and Methods. In silico docking was performed using PatchDock and FireDock online server http://bioinfo3d.cs.tau.ac.il/PatchDock/ .

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Silico

Journal: Oncogene

Article Title: Galectin-3 regulates p21 stability in human prostate cancer cells

doi: 10.1038/onc.2012.528

Figure Lengend Snippet: Proposed model for the regulation of p21 by Gal-3 in p53 expressing prostate cancer cells. Gal-3 binds to p21 through CRD, which can be blocked by sugars i.e. lactose or GCS-100. The interaction between Gal-3 and p21 promotes p21 stability, and the regulated p21 partially mediates Gal-3 functions associated with cell proliferation, apoptosis, and invasion. Acting as a transcription factor, p53 positively regulates p21 and negatively regulates Gal-3.

Article Snippet: Monoclonal rat anti-Gal-3 antibody was isolated from the supernatant of hybridoma (catalog number: TIB-166, American Type Culture Collection, Manassas, VA, USA); Customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Grand Island, NY, USA); mouse anti-p21 was purchased from BD Biosciences (San Jose, CA, USA); cisplatin, thiazolyl blue tetrazolium bromide (MTT), cycloheximide, mouse anti- β- actin was purchased from Sigma Chemicals (St. Louis, MO, USA); rabbit anti-caspase-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing