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

Journal: The Journal of Clinical Investigation

Article Title: Chronic mTOR activation induces a degradative smooth muscle cell phenotype

doi: 10.1172/JCI131048

Figure Lengend Snippet: Ascending aortas were procured from 24 subjects undergoing aortic surgery (Aneurysm) or from organ donors (Nondilated). (A and B) Immunofluorescence analysis for CD45 (red), SMMHC (green), LAMP2 (also known as Mac-3; white), and overlays with DAPI-labeled nuclei (blue) showing LAMP2+ leukocytes (arrows) in the intima (I) and LAMP2+ SMCs (arrows) in the media (M); inset of spleen positive control (scale bars: 100 μm). (C) Expression of SMMHC and LAMP2 (n = 12) and correlation of SMMHC to LAMP2 (n = 24). px2, pixels squared. (D) Verhoeff stain of aortic media for elastin (scale bar: 200 μm). (E) Elastin expression (n = 12) and correlation of elastin loss to LAMP2 (n = 24). (F) H&E stain of media (scale bar: 200 μm). (G) Number of medial cells per cross section (cs) (n = 12) and correlation of medial cells to LAMP2 (n = 24). (H) Correlation of media thickness and aorta diameter to LAMP2 (n = 24). Data are represented as individual values with mean ± SEM bars or linear regression lines. *P < 0.05, **P < 0.01 by t test (C, E, and G) or Spearman’s test for r correlation coefficient (C, E, G, and H).

Article Snippet: Frozen sections were labeled with phalloidin (Thermo Fisher Scientific) or an antibody against MMP2 (catalog ab37150, Abcam), mouse LAMP2 (catalog 1921-01, SouthernBiotech), GAL3 (catalog CL8942AP, Cedarlane), β-catenin (catalog 610154, BD Biosciences), MITF (catalog MABE78, Millipore), p-S6 (catalog 4858, Cell Signaling Technology), mouse CD45 (catalog 550539, BD Biosciences), CD11b (catalog 550282, BD Biosciences), CD68 (catalog MCA 1957, Bio-Rad), F4/80 (catalog 14-4801-81, Thermo Fisher Scientific), MOMA-2 (catalog MAB1852, Millipore), TER-119 (catalog 116212, BioLegend), mouse and human SMMHC (catalog ab53219, Abcam), human CD45 (catalog MA5-17687, Thermo Fisher Scientific), and human LAMP2 (catalog ab25631, Abcam) or isotype-matched, irrelevant IgG.

Techniques: Immunofluorescence, Labeling, Positive Control, Expressing, Staining

Journal: The Journal of Clinical Investigation

Article Title: Chronic mTOR activation induces a degradative smooth muscle cell phenotype

doi: 10.1172/JCI131048

Figure Lengend Snippet: Myh11-CreERT2 mT/mG (Tsc1+/+) and Tsc1fl/fl Myh11-CreERT2 mT/mG (Tsc1−/−) mice were treated with tamoxifen at 1.5 weeks and the thoracic aortas were analyzed at 24 weeks. (A) Selected transcript expression by bulk RNA-seq. (B and C) Protein expression by Western blot (n = 8). (D and E) β-Catenin and MITF expression (white) with RFP (red), GFP (green), and DAPI (blue) overlay (scale bars: 100 μm), and quantified as medial percentage area (n = 5–7). Alternatively, cultured GFP+ SMCs were analyzed by P3. (F) Expression of signaling molecules and lysosomal membrane proteins after treatment with rapamycin (RAPA) at 0–100 ng/mL for 6 days in serum-supplemented medium. (G and H) Effects of Ctnnb1 and Mitf knockdown by siRNA versus control (Cntrl) for 3 days. (I) Fold inhibition after rapamycin or siRNA treatment relative to control treatment (dotted lines) in Tsc1−/− cells (n = 3–5). Data are represented as individual values with mean ± SEM bars or as box-and-whisker plots with interquartile range, median, minimum, and maximum. *P < 0.05; **P < 0.01; ***P < 0.001; FDR‑adjusted P values (A), 2-way ANOVA (B and I), or t test (D and E).

Article Snippet: Frozen sections were labeled with phalloidin (Thermo Fisher Scientific) or an antibody against MMP2 (catalog ab37150, Abcam), mouse LAMP2 (catalog 1921-01, SouthernBiotech), GAL3 (catalog CL8942AP, Cedarlane), β-catenin (catalog 610154, BD Biosciences), MITF (catalog MABE78, Millipore), p-S6 (catalog 4858, Cell Signaling Technology), mouse CD45 (catalog 550539, BD Biosciences), CD11b (catalog 550282, BD Biosciences), CD68 (catalog MCA 1957, Bio-Rad), F4/80 (catalog 14-4801-81, Thermo Fisher Scientific), MOMA-2 (catalog MAB1852, Millipore), TER-119 (catalog 116212, BioLegend), mouse and human SMMHC (catalog ab53219, Abcam), human CD45 (catalog MA5-17687, Thermo Fisher Scientific), and human LAMP2 (catalog ab25631, Abcam) or isotype-matched, irrelevant IgG.

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Cell Culture, Inhibition, Whisker Assay