Journal: Cell Death & Disease

Article Title: High cholesterol triggers white matter alterations and cognitive deficits in a mouse model of cerebrovascular disease: benefits of simvastatin

doi: 10.1038/s41419-018-1199-0

Figure Lengend Snippet: Cresyl-violet stained brain sections show the targeted areas (dashed line) of the corpus callosum (cc, a ) and internal capsule (IC, b ) used for quantitative analysis (top right panels, respectively). In TGF mice, galectin-3 (Gal-3)-immunofluorescent (green, Cy2) cell number ( a ) or surface area ( b ) was dramatically increased by HCD (arrows, right panels, respectively), an increase that was significantly reduced by SV treatment in both regions, and not seen in HCD-treated WT mice. Cpu caudate putamen, cg cingulate cortex, LV lateral ventricle, fi fibria. Bars: 200 μm (cc) and 300 μm (IC), * p < 0.05; *** p < 0.001

Article Snippet: For single immunofluorescent staining, free-floating freezing sections were incubated overnight with either goat anti-collagen IV (Millipore, Billerica, USA, catalog # AB769, 1:300, for staining brain vessels) or anti-doublecortin (DCX- (C-18), Santa Cruz, catalog # sc-8066, 1:1000, a marker of migrating neuroblasts), rabbit anti-GFAP (Dako Canada, Burlington, Code-Nr. Z 0334, 1:2,000, a marker of astrocytes), anti-phospho MAP kinase (Erk 1/2, Cell signaling, catalog # 9101, 1:100), or anti-oligodendrocyte transcription factor 2 (Olig2, Millipore, catalog # AB9610, 1:1000, a marker of oligodendrocytes), rat anti-galectin-3 (Gal-3) (Mac-2, Cedarlane, catalog # CL8942AP, Burlington, ON, Canada, 1:1500, a marker for active WM-related microglial cells) or anti-F4/80 (Bio-Rad, catalog # MCA497, Mississauga, ON, Canada, 1:100, a marker for active microglia or macrophages), or mouse anti-polysialic acid-neural cell adhesion molecule (PSA-NCAM, Millipore, catalog # MAB5324, 1:800, protein that helps migration of oligodendrocyte precursor cells (OPC)) followed by incubation with the corresponding cyanin 2 (Cy2, green)-, Alexa 488 (green)-, cyanin 3 (Cy3, red)- or Alexa 594 (red)-conjugated secondary antibodies (Jackson laboratory, West Grove, PA, USA).

Techniques: Staining

Journal: Oncotarget

Article Title: Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells

doi:

Figure Lengend Snippet: A, Western blot analysis shows Gal-3 protein expression in Nthy-ori 3-1, TPC1 and FTC-133 cells. β-actin was used as the loading control. B, Gal-3 knockdown led to decreased cell growth of FTC-133 cells. Cell growth was analyzed by MTT assay. The value of day 1 was set as 1. ** P < 0.01 vs vector. Points in MTT assay represent the mean of three independent experiments; bars , SE. C, FTC-133 and TPC1 stable cells were established as in Material and Methods. They were either untreated or treated with 1 μM DXR for 24 hours. Cell viability was determined by MTT. The value of untreated cells was set as 1. **P < 0.01, *P < 0.05. Columns represent the mean of three independent experiments; bars, SE. D, TPC1 cells were exposed to the indicated concentration of CDDP or DXR. Cell lysates were prepared and processed for western blot assay 24 hours after treatment. E, TPC1 cells were treated with 0.5 μM of DXR. Kinetic analyses of the indicated proteins were done at the times indicated. Analysis was normalized by β-actin. Points represent the mean of two independent experiments; bars , SE.

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); customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Carlsbad, CA); mouse anti-V5 was purchased from Invitrogen; polyclonal rabbit anti-Bax, mouse anti-Bcl-xl, mouse anti-p-ERK, mouse ERK1/2 and rabbit anti-p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-β-actin was purchased from Sigma-Aldrich; monoclonal mouse anti-Bax was purchased from BD Biosciences (San Diego, CA); mouse anti-p21, rabbit anti-Bcl2, rabbit anti-PARP (9542L), rabbit anti-cleaved caspase-3, rabbit anti-Akt and mouse anti-caspase-8 were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA);

Techniques: Western Blot, Expressing, MTT Assay, Plasmid Preparation, Concentration Assay

Journal: Oncotarget

Article Title: Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells

doi:

Figure Lengend Snippet: A, TPC1 cells were transfected with si-Control or si-Gal-3 for 24 hours, and then either untreated or treated with 1 μM of DXR for 24 hours. The release of cytochrome c was determined in the cytoplasmic fraction of TPC1 cells at the times indicated. The quantification of band intensity was performed using ImageJ software and the value of si-Control for 0 hour was set as 1. B and C, TPC1 cells were treated as in A. Indicated protein expressions were determined. β-actin was used as the loading control. D, TPC1 cells were pretreated with 1% of GCS-100/MCP for 3 hours, and then either left untreated or treated with 1 μM DXR for 24 hours. (Upper) Cell lysates were isolated, and PARP cleavage levels or cleaved caspase-3 expression were determined. (Lower) Bax oligomerization assay. Cells were cross-linked with 1, 6-bismaleimidohexane (BMH) and immunoblotted with polyclonal anti-Bax antibody. NS means non-specific band. E, TPC1 cells were transfected with indicated siRNA for 24 hours, and then either untreated (white columns) or treated with 0.5 μM of DXR (black columns) for 24 hours. Western blot analysis shows Gal-3 and Bax protein expression. Cell viability was analyzed by MTT assay. The value of untreated control cells was set as 1. The symbols of a, b, c and d indicate the black columns of lane 2, 4, 6 and 8 in the graph, respectively. ** P < 0.01, * P < 0.05. Columns represent the mean of three independent experiments; bars , SE.

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); customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Carlsbad, CA); mouse anti-V5 was purchased from Invitrogen; polyclonal rabbit anti-Bax, mouse anti-Bcl-xl, mouse anti-p-ERK, mouse ERK1/2 and rabbit anti-p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-β-actin was purchased from Sigma-Aldrich; monoclonal mouse anti-Bax was purchased from BD Biosciences (San Diego, CA); mouse anti-p21, rabbit anti-Bcl2, rabbit anti-PARP (9542L), rabbit anti-cleaved caspase-3, rabbit anti-Akt and mouse anti-caspase-8 were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA);

Techniques: Transfection, Software, Isolation, Expressing, Western Blot, MTT Assay

Journal: Oncotarget

Article Title: Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells

doi:

Figure Lengend Snippet: A and B, Co-immunoprecipitation assay. TPC1 cells were treated with 0.5 μM DXR for 24 hours. Cell lysates were immunoprecipitaed with IgG rabbit, polyclonal anti-Bax, or polyclonal anti-Gal-3 antibody. The immunoprecipitates and input lysates were analyzed by immunoblotting with indicated antibodies. Input lysates indicate lysates used for immunoprecipitation from TPC1 cells and were used as positive control. C, TPC1 cells were pretreated with 1% of GCS-100/MCP for 3 hours, and then either left untreated or treated with 1 μM DXR for 24 hours. Cell lysates were immunoprecipitated with polyclonal anti-Gal-3 antibody. The immunoprecipitates and input lysates were analyzed by immunoblotting with indicated antibodies. D, Co-localization of Gal-3 and Bax in TPC1 cells treated with 0.5 μM DXR for 24 hours. TPC1 cells were immunofluorescently labelled with anti-Gal-3 (red), anti-Bax (green) antibodies and Hoechst 33258 (nuclear stain, blue). Scale bar represents 50 μm. E, Prediction of the interaction of Gal-3 carbohydrate recognition domain (CRD) with Bax. The references about the structure of Gal-3 CRD and Bax were indicated in Materials and Methods. In silico docking was performed using Second ClusPro 2.0 server ( http://cluspro.bu.edu/login.php ). Asn means asparagine.

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); customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Carlsbad, CA); mouse anti-V5 was purchased from Invitrogen; polyclonal rabbit anti-Bax, mouse anti-Bcl-xl, mouse anti-p-ERK, mouse ERK1/2 and rabbit anti-p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-β-actin was purchased from Sigma-Aldrich; monoclonal mouse anti-Bax was purchased from BD Biosciences (San Diego, CA); mouse anti-p21, rabbit anti-Bcl2, rabbit anti-PARP (9542L), rabbit anti-cleaved caspase-3, rabbit anti-Akt and mouse anti-caspase-8 were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA);

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Positive Control, Staining, In Silico

Journal: Oncotarget

Article Title: Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells

doi:

Figure Lengend Snippet: A, Gal-3 is composed of three structural domains: (a) a NH2-terminal domain of 12 amino acids; (b) a repeated collagen-like sequence rich in glycine, proline, and tyrosine; and (c) a COOH-terminal CRD. The C-terminal domain includes the NWGR motif. Wild type Gal-3 and mutant Gal-3 G182A were generated and cloned into the pcDNA6/V5 expression vector. B, Indicated plasmids were transiently transfected into 293 cells. After 48 hours, cell lysates were immunoprecipitaed with anti-V5 antibody. The immunoprecipitates and input lysates were analyzed by immunoblotting with indicated antibodies. Input lysates from 293 cells were used as positive control. C, Western blot analysis shows Gal-3 and V5 protein expression in TPC1 stable clones. Wild type Gal-3 and mutant Gal-3 G182A in pcDNA6/V5 expression vector were used for establishment of stable clones (vector control; VC, wild type Gal-3; WT and mutant Gal-3 G182A; #4 and #7). β-actin was used as the loading control. D, TPC1 stable cells were either untreated or treated with 1 μM DXR for 24 hours. PARP cleavage levels and cleaved caspase-3 expression were determined. β-actin was used as the loading control. E, TPC1 stable cells were treated as in D. Cell viability was analyzed by MTT assay. Cell viability of treated cells was represented as the percentage of untreated control cells. ** P < 0.01 vs untreated cells. Columns represent the mean of three independent experiments; bars , SE.

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); customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Carlsbad, CA); mouse anti-V5 was purchased from Invitrogen; polyclonal rabbit anti-Bax, mouse anti-Bcl-xl, mouse anti-p-ERK, mouse ERK1/2 and rabbit anti-p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-β-actin was purchased from Sigma-Aldrich; monoclonal mouse anti-Bax was purchased from BD Biosciences (San Diego, CA); mouse anti-p21, rabbit anti-Bcl2, rabbit anti-PARP (9542L), rabbit anti-cleaved caspase-3, rabbit anti-Akt and mouse anti-caspase-8 were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA);

Techniques: Sequencing, Mutagenesis, Generated, Clone Assay, Expressing, Plasmid Preparation, Transfection, Western Blot, Positive Control, MTT Assay

Journal: Oncotarget

Article Title: Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells

doi:

Figure Lengend Snippet: A, TPC1, HeLa, HT1080 and PC3 cells were pretreated with 1% of GCS-100/MCP for 3 hours, and then either left untreated or treated with 1 μM DXR for 24 hours. Cell viability was determined by MTT. The value of untreated cells was set as 1. * P < 0.05. Columns represent the mean of three independent experiments; bars , SE. B, Schematic representation for the regulation of apoptosis of Gal-3 by binding to Bax. Gal-3 heterodimerizes Bax, mediated by CRD of Gal-3, leading to anti-apoptotic characteristic. Gal-3/Bax interaction was suppressed by sugar antagonist of Gal-3, in which in turn cells became sensitive to apoptosis.

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); customized polyclonal rabbit anti-Gal-3 antibody was created by Invitrogen (Carlsbad, CA); mouse anti-V5 was purchased from Invitrogen; polyclonal rabbit anti-Bax, mouse anti-Bcl-xl, mouse anti-p-ERK, mouse ERK1/2 and rabbit anti-p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-β-actin was purchased from Sigma-Aldrich; monoclonal mouse anti-Bax was purchased from BD Biosciences (San Diego, CA); mouse anti-p21, rabbit anti-Bcl2, rabbit anti-PARP (9542L), rabbit anti-cleaved caspase-3, rabbit anti-Akt and mouse anti-caspase-8 were purchased from Cell Signaling Technology (Beverly, MA); purified rabbit IgG was purchased from ZYMED (San Francisco, CA);

Techniques: Binding Assay

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: PLoS ONE

Article Title: The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

doi: 10.1371/journal.pone.0020665

Figure Lengend Snippet: (A) Western blot analysis, using antibodies to HIPK2 and Gal-3, performed on TCE obtained from PTC-derived K1 cell line stably transfected with pSUPER.retro control vectors (ctr) and pSUPER-HIPK2 interfering construct (HIPK2i). (B) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (A). (C) Western blot analysis of HIPK2 and Gal-3 in K1 cell transiently transfected with control vector pCMV-FLAG (Flag) and with pCMV-FLAG-HIPK2 (Flag-HIPK2) expression construct. (D) Inverted images of agarose gels showing semiquantitative RT-PCR analyses of HIPK2 and Gal-3 gene expression performed on total RNA extracted from K1 cells described in (C). (E) Western blot analysis of HIPK2, p53Ser46, total p53 and Gal-3 in K1 cell transiently transfected with the kinase-dead pEGFP-HIPK2 K221R (GFP-HIPK2 K221R ) and the wild type pEGFP-HIPK2 (GFP-HIPK2) HIPK2 expression constructs. Western blot experiments were normalized using β-actin protein expression while the 18S gene was amplified as control for semiquantitative RT-PCR analyses.

Article Snippet: The following antibodies were used in immunoblotting: rabbit anti-HIPK2 antiserum (kindly provided by L. Schmitz), purified rat mAb anti-Gal-3 antibody (Mabtech AB, Nacka Strand, Sweden), mouse mAb anti-p53 (Santa Cruz), mouse mAb anti-p53Ser46 (Cell Signaling Technology) and mouse mAb anti-β-actin (Sigma), and HRP-conjugated anti-rabbit, anti-rat or anti-mouse, antibodies (Sigma).

Techniques: Western Blot, Derivative Assay, Stable Transfection, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Amplification