goat anti galectin 4  (R&D Systems)

Journal: Molecular Oncology

Article Title: Glycosylated LGALS3BP is highly secreted by bladder cancer cells and represents a novel urinary disease biomarker

doi: 10.1002/1878-0261.70140

Figure Lengend Snippet: LGALS3BP is highly expressed in bladder cancer tissues. (A) Immunohistochemical analysis of LGALS3BP expression in bladder cancer tissues at first transurethral resection of the bladder tumor (TURBT). Representative images of non‐muscle‐invasive bladder cancer (NMIBC) and muscle‐invasive bladder cancer (MIBC) tissue samples are shown. Scale bar: 250 μ m . NMIBC n = 18; MIBC n = 11. (B) Contingence table reporting analysis of LGALS3BP expression in patients who progressed (MIBC, n = 11) or not (NMIBC, n = 18). Patients were stratified based on expression of LGALS3BP (high or low) and progression (NMIBC vs . MIBC).

Article Snippet: Formalin‐fixed, paraffin‐embedded tissue sections were immunostained using a goat polyclonal antibody against human LGALS3BP (1 : 400 dilution; 60‐min incubation; catalog no. AF2226; R&D Systems, Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Molecular Oncology

Article Title: Glycosylated LGALS3BP is highly secreted by bladder cancer cells and represents a novel urinary disease biomarker

doi: 10.1002/1878-0261.70140

Figure Lengend Snippet: LGALS3BP expression in bladder cancer cell lines. (A) Secreted and (B) intracellular LGALS3BP protein levels in a panel of human bladder cancer cell lines analyzed through western blotting in cell culture supernatants ( n = 2) and cell lysates ( n = 2), respectively. Equal amounts of samples were loaded per lane. GAPDH was used as a loading control. (C) Histogram showing secreted LGALS3BP levels (ng·mL −1 ) evaluated by a 1959‐based sandwich enzyme‐linked immunosorbent assay (ELISA) ( n = 3). Data are presented as mean ± standard deviation (SD). In vitro IC 50 curves evaluated by 3‐(4,5‐dimethyldiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assays by exposing (D) T‐24 ( n = 2) and (E) SW780 ( n = 3) cells for 72 h to increasing doses of Cisplatin and DM4. IC 50 values are reported for each cell line considered upon different treatments. Data are presented as mean ± SD.

Article Snippet: Formalin‐fixed, paraffin‐embedded tissue sections were immunostained using a goat polyclonal antibody against human LGALS3BP (1 : 400 dilution; 60‐min incubation; catalog no. AF2226; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Cell Culture, Control, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Standard Deviation, In Vitro

Journal: Molecular Oncology

Article Title: Glycosylated LGALS3BP is highly secreted by bladder cancer cells and represents a novel urinary disease biomarker

doi: 10.1002/1878-0261.70140

Figure Lengend Snippet: 1959‐sss/DM4 antitumor activity in subcutaneous xenograft models of bladder cancer cells. (A) Single animal tumor growth expressed as tumor volume (m m 3 ) of T‐24 subcutaneous xenografts in nude mice treated with vehicle (PBS) ( n = 2) or 1959‐sss/DM4 (10 mg·kg −1 ; twice weekly; three total i.v. injections) ( n = 2). Arrows indicate treatment administration. Data are presented as mean ± standard error of the mean (SEM). (B) Tumor growth expressed as mean tumor volume (m m 3 ) from the first treatment of SW780 subcutaneous xenografts in nude mice treated with vehicle ( n = 6) or 1959‐sss/DM4 ( n = 6). Arrows indicate treatment administration (10 mg·kg −1 ; twice weekly; three total i.v. injections). Data are presented as mean ± SEM. (C) Tumor weight (g) at the endpoint of the SW780‐xenograft in vivo therapeutic experiment (left panel). Statistical significance was determined by t ‐test (** P < 0.01). Commercial anti‐LGALS3BP ELISA kit showing circulating LGALS3BP levels (ng·mL −1 ) in serum derived from SW780‐xenografted mice treated with PBS or 1959‐sss/DM4 (middle panel). Correlation between circulating LGALS3BP levels (ng·mL −1 ) in PBS‐treated SW780‐xenografted mice serum and corresponding tumor volumes (m m 3 ) (right panel). Correlation value is reported ( R 2 = 0.8399). (D) Representative immunohistochemistry (IHC) sections of SW780 tumors in mice treated with PBS ( n = 6) or 1959‐sss/DM4 ( n = 6) stained for LGALS3BP. Arrows indicate giant cells, which are the hallmarks of mitotic catastrophe. * P < 0.05. Magnification 40X. Scale bar: 50 μ m .

Article Snippet: Formalin‐fixed, paraffin‐embedded tissue sections were immunostained using a goat polyclonal antibody against human LGALS3BP (1 : 400 dilution; 60‐min incubation; catalog no. AF2226; R&D Systems, Minneapolis, MN, USA).

Techniques: Activity Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Derivative Assay, Immunohistochemistry, Staining

Journal: Molecular Oncology

Article Title: Glycosylated LGALS3BP is highly secreted by bladder cancer cells and represents a novel urinary disease biomarker

doi: 10.1002/1878-0261.70140

Figure Lengend Snippet: Increased Urinary LGALS3BP Expression in Bladder Cancer Patients. (A) Biotinylated 1959‐based sandwich ELISA showing LGALS3BP expression levels in urine from healthy donors (HD) ( n = 47) or bladder cancer patients (BC) ( n = 26) samples of a first cohort of patients. Statistical significance was obtained by using the Mann–Whitney test (**** P < 0.0001). Data are presented as distribution with dotted lines corresponding to the 25th percentile, the median, and the 75th percentile from bottom to top in the violin plot. (B) Biotinylated 1959‐based sandwich ELISA showing LGALS3BP urinary expression levels (ng·mL −1 ) in HD ( n = 6), BC low‐grade ( n = 26), or BC high‐grade ( n = 12) of a second cohort of patients. Statistical significance was obtained by using the Kruskal–Wallis test (** P < 0.01; * P < 0.05). Data are presented as distribution with dotted lines corresponding to the 25th percentile, the median, and the 75th percentile from bottom to top in the violin plot. (C) Biotinylated 1959‐based sandwich ELISA showing LGALS3BP urinary expression levels (ng·mL −1 ) in HD ( n = 53) or NMIBC ( n = 60) patients by pooling data from the two previously analyzed patient cohorts. Statistical significance was obtained by using the Mann–Whitney test (**** P < 0.0001). Data are presented as distribution with dotted lines corresponding to the 25th percentile, the median, and the 75th percentile from bottom to top in the violin plot. (D) ROC curve generated by pooling data from the two previously analyzed patient cohorts. (E) Western blotting image of LGALS3BP protein pattern in urine samples from HD or BC patients. Equal amounts of samples were loaded per lane. Urinary LGALS3BP levels (ng·mL −1 ) detected through the biotinylated 1959‐based sandwich ELISA are reported in the corresponding healthy donors and BC patient samples. n = 2.

Article Snippet: Formalin‐fixed, paraffin‐embedded tissue sections were immunostained using a goat polyclonal antibody against human LGALS3BP (1 : 400 dilution; 60‐min incubation; catalog no. AF2226; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Sandwich ELISA, MANN-WHITNEY, Generated, Western Blot

Journal: Molecular Oncology

Article Title: Glycosylated LGALS3BP is highly secreted by bladder cancer cells and represents a novel urinary disease biomarker

doi: 10.1002/1878-0261.70140

Figure Lengend Snippet: LGALS3BP glycosylation. (A) Western blot images of LGALS3BP protein pattern in cell lysates and cell culture supernatants of T‐24 cells treated or not with OSMI‐1 (50 μ m ), tunicamycin (TUN) (10μg·mL −1 ), or Kifunensine (KIF) (5 μ m ). Molecular weight markers are indicated on the left (kDa). n = 2. (B) 1959‐based sandwich ELISA ( n = 2) and (C) commercial anti‐LGALS3BP ELISA kit ( n = 2) for the detection of secreted LGALS3BP levels in cell culture supernatants of T‐24 cells under different treatment conditions (control, OSMI‐1, TUN, KIF). The bar graph shows normalized values expressed in arbitrary units, with all conditions normalized to the control, which is set to 1. (D) Representative confocal microscopy images of T‐24 cells under two conditions: vehicle (left) and treated with 5 μ m KIF (right). Nuclei are stained in blue (DAPI), and the green signal represents LGALS3BP. Scale bar: 20 μ m . Images are slices merged from z‐stack acquisition, n = 2. (E) Histogram representing the mean fluorescence intensity (MFI) of LGALS3BP signal per nucleus measured by confocal microscopy in T‐24 cells treated or not with KIF 5 μ m (unpaired t ‐test, * P < 0.05). n = 2. Data are presented as mean ± standard deviation (SD).

Article Snippet: Formalin‐fixed, paraffin‐embedded tissue sections were immunostained using a goat polyclonal antibody against human LGALS3BP (1 : 400 dilution; 60‐min incubation; catalog no. AF2226; R&D Systems, Minneapolis, MN, USA).

Techniques: Glycoproteomics, Western Blot, Cell Culture, Molecular Weight, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Control, Confocal Microscopy, Staining, Fluorescence, Standard Deviation