Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: PCR primer sequences and NCBI accession numbers for mucin genes/transcripts. The letter P denotes sequences that are predicted.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques:

Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: The peripheral cornea expresses MUC1, MUC4 and MUC16 at higher levels than the central cornea. Epithelium was debrided from both the peripheral (~75% corneal surface area) and central (~25% corneal surface area) cornea and quantitative PCR was performed. In all species examined (A-rhesus macaque, B-canine, C-rabbit), mucin expression tended to be higher in the peripheral cornea when compared with the central cornea. Data represents relative mucin expression normalized to the respective housekeeping gene per species (Table 1, n = 6 eyes per species from different individuals). Error bars represent standard error of the mean. Asterisk indicates p ≤ 0.05.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: Corneal mucin mRNA transcript expression pattern differs between species. Quantitative PCR of mucin mRNA transcript expression in humans (A), rhesus macaques (B), dogs (C) and rabbits (D). Similar patterns of mucin mRNA expression existed in humans, rhesus macaques and rabbits with MUC16 being expressed to the highest levels, followed by MUC1 and MUC4 expression being the lowest detected. The rabbit had a unique pattern of mucin mRNA expression with MUC4 being expressed at the highest level, MUC1 and MUC16 being expressed at lower levels. Data represents relative mucin expression normalized to the respective housekeeping gene per species (Table 1, n = 5 eyes per species from different individuals). Error bars represent standard error of the mean.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Experimental eye research

Article Title: Species variation and spatial differences in mucin expression from corneal epithelial cells

doi: 10.1016/j.exer.2016.09.001

Figure Lengend Snippet: MUC16 glycoprotein was detectable in human, rhesus macaque and canine corneal epithelial extracts. Using a human-specific MUC16 antibody (OC125), large high molecular weight protein was detected a human, rhesus macaque and canine corneal epithelial extracts, whereas no protein was detected in rabbit epithelial extracts.

Article Snippet: Human MUC16 , Hs01065189_m1 , , {"type":"entrez-nucleotide","attrs":{"text":"NM_024690","term_id":"83367076","term_text":"NM_024690"}} NM_024690.

Techniques: High Molecular Weight

Journal: PLOS ONE

Article Title: NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

doi: 10.1371/journal.pone.0285161

Figure Lengend Snippet: Antibodies were screened via ELISA for binding to immobilized MSLN and MUC16/CA125 proteins. Human serum albumin (HSA) was used as negative control. Panel A shows that the antibody referred to herein as NAV-001 was the only antibody from the group that did not bind to MUC16/CA125 protein (P = 0.000025). To determine if MUC16/CA125 perturbs anti-MSLN binding to MSLN protein, immobilized MSLN was probed with each antibody with or without MUC16/CA125 (CA). As shown in panel B, MUC16/CA125 had no impact on antibodies binding to MSLN. All data represent a minimum of triplicate experiments.

Article Snippet: To confirm MSLN and MUC16/CA125 expression in PDFs, we employed IHC using a rabbit anti-MSLN antibody that binds the same epitope as NAV-001 and a commercial rabbit anti-MUC16/CA125 antibody (Novus), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control

Journal: PLOS ONE

Article Title: NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

doi: 10.1371/journal.pone.0285161

Figure Lengend Snippet: Antibodies Ab-1, Ab-2 and NAV-001 were converted into SN-38 ADCs and tested for cytotoxicity using the isogenic MSLN-expressing OVCAR-3 and OV-KD cells, the latter which lacks MUC16/CA125 expression. As shown in Panel A, Ab-1 and Ab-2 had reduced OVCAR-3 target cell (CA125 + ) killing as compared to NAV-001 (P < 0.00011). All three ADCs killed the isogenic OV-KD cells (CA125 - ) at a similar magnitude confirming the negative effect of MUC16/CA125 on ADCs containing antibodies that bind it. Shown is the differential killing of each ADC when cultures were treated with 50 ng/mL of each ADC. Panel B is a schematic representation of the potential mechanism by which MUC16/CA125 suppresses ADC killing through antibody binding and reduced internalization. All data represent a minimum of triplicate experiments.

Article Snippet: To confirm MSLN and MUC16/CA125 expression in PDFs, we employed IHC using a rabbit anti-MSLN antibody that binds the same epitope as NAV-001 and a commercial rabbit anti-MUC16/CA125 antibody (Novus), respectively.

Techniques: Expressing, Binding Assay

Journal: PLOS ONE

Article Title: NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

doi: 10.1371/journal.pone.0285161

Figure Lengend Snippet: Antibody 2 (Ab-2) and NAV-001 were converted into ADCs by linking the topoisomerase II cytotoxin PNU-159682 and both were tested for cytotoxicity against the isogenic MSLN-expressing OVCAR-3 (panel A and C) and OV-KD (panel B and D) cell lines. As shown in Panels A and B, a similar effect of target cell killing was observed as the SN-38 formatted ADCs shown in , whereby the MUC16/CA125 binding Ab-2-PNU had reduced killing in OVCAR-3 (A) as compared to NAV-001-PNU(P < 0.0033), while similar killing was observed for both against the isogenic non-MUC16/CA125-expressing OV-KD cells (B). Antibodies were tested for cellular uptake using the same cell pairs and as shown in Panels C (OVCAR-3) and D (OV-KD), Ab-2 has reduced internalization as compared to NAV-001 in OVCAR-3 cells (P < 0.00008) but similar internalization kinetics in the OV-KD cells. All data represent a minimum of triplicate experiments.

Article Snippet: To confirm MSLN and MUC16/CA125 expression in PDFs, we employed IHC using a rabbit anti-MSLN antibody that binds the same epitope as NAV-001 and a commercial rabbit anti-MUC16/CA125 antibody (Novus), respectively.

Techniques: Expressing, Binding Assay

Journal: PLOS ONE

Article Title: NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

doi: 10.1371/journal.pone.0285161

Figure Lengend Snippet: Panel 6A, PDFs used for each study were confirmed for MSLN and MUC16/CA125 expression by IHC analyses. As shown, all tumors had robust homogeneous MSLN staining, while the mesothelioma and TNBC tumors also expressed MUC16/CA125. These tumors served as models to confirm NAV-001-PNU in vivo efficacy against MUC16/CA125 expressing tumors. Panel B, tumor growth and tolerability of NAV-001 in PDX models. As shown, single-dose of NAV-001 at 0.25 or 0.75 mg/kg showed significant anti-tumor efficacy (P < 0.005), and long-term regression after 0.75 mg/kg single-dose regimen. Triangles represent day of dosing post randomization. Each cohort of each study employed 6 mice.

Article Snippet: To confirm MSLN and MUC16/CA125 expression in PDFs, we employed IHC using a rabbit anti-MSLN antibody that binds the same epitope as NAV-001 and a commercial rabbit anti-MUC16/CA125 antibody (Novus), respectively.

Techniques: Expressing, Staining, In Vivo

Journal: Journal of Cancer

Article Title: Tranexamic acid reduces endometrial cancer effects through the production of angiostatin

doi: 10.7150/jca.68169

Figure Lengend Snippet: Effects of TA treatment on plasminogen, plasmin and tPA levels. Thirty weeks after the start of the study, by western blotting, the expression of plasminogen (A) , plasmin (B) and tPA (C) in uterus on MNU- and estradiol-induced endometrial cancer model mice were measured. By ELISA kit, the levels of plasminogen (D) , plasmin (E) and tPA (F) in plasma on MNU- and estradiol-induced endometrial cancer model mice were measured. Values are expressed as the mean ± standard deviation (SD) derived from 10 animals. * p < 0.05. TA: tranexamic acid, tPA: tissue plasminogen activator, MNU: N -methyl- N -nitrosourea.

Article Snippet: The plasma levels of carbohydrate antigen 125 (CA125), interleukin (IL)-6, tumor necrosis factor (TNF-α), plasminogen, plasmin, and tPA were determined using commercial ELISA kits (CA125: Bioassay Technology Laboratory, Shanghai, China; Il-6: Proteintech, Rosemont, IL, USA; TNF-α: R&D Systems, Minneapolis, MN, USA; plasminogen and plasmin: LSBio, Seattle, WA, USA; tPA: Abcam, Cambridge, UK) according to the respective manufacturers' instructions.

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Standard Deviation, Derivative Assay

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: (A) hTERT-HPNE, HPDE, PANC-1, SW1990, and Pa03C cells were seeded in 96-well plates pre-coated with either mesothelin (MSLN) or BSA (control). After 30 min of incubation, the relative number of adherent cells to MSLN- versus BSA-coated wells was quantified by WST-1 assay. Data represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to control. (B) Representative flow cytometric histograms of MUC16 surface expression on hTERT-HPNE, HPDE, PANC-1, SW1990, and Pa03C cells using indirect single-color immunofluorescence and an anti-MUC16 mAb or an isotype control. (C) MUC16-overexpressing SW1990 or Pa03C cells were pre-incubated with either a function-blocking anti-MUC16 mAb M11 (20 μg/ml) or an isotype control for 1 h at 37°C, and then used in the cell binding assay as described in (A). (D) Scramble control or MUC16-KD SW1990 and Pa03C cells were seeded in 96-well plates pre-coated with MSLN or BSA (control). After 30 min of incubation, adherent cells were quantified by WST-1 assay. Data represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to MUC16-KD cells.

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Incubation, WST-1 Assay, Expressing, Immunofluorescence, Blocking Assay, Cell Binding Assay

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: Whole lysates from scramble control (A) or MUC16-KD (B) SW1990 and Pa03C cells were immunoprecipitated with either an anti-MUC16 or anti-MSLN or an isotype control antibody, and analyzed for MSLN or MUC16 reactivity by immunoblotting. Lysates from SW1990 and Pa03C cells were also analyzed as input controls.

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: hTERT-HPNE, HPDE, PANC-1, SW1990, and Pa03C cells were starved in serum-free medium and incubated in the presence or absence of MSLN (1 μg/ml) overnight. (A) MMP-7, (B) MMP-2, and (C) MMP-9 mRNA levels were then determined by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least three independent experiments. *, p < 0.01 with respect to corresponding control. (D) MUC16, MSLN, and MMP-7 expression in pancreatic cancer tissue specimens was determined by immunohistochemical staining using an anti-MUC16, anti-MSLN, or anti-MMP-7 antibody, respectively. Strong immunostaining was detected in ∼ 90% of cancerous tissues, whereas no immunoreactivity was present in the ducts or acini of normal pancreas tissue.

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Incubation, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Immunostaining

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: (A) SW1990 cells were starved in serum-free medium overnight and incubated with MSLN (1 μg/ml) for the indicated periods of time. The mRNA levels of MMP-7 were quantified by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least three independent experiments. *, p < 0.05 with respect to cells treated with MSLN (1 μg/ml) for 0 h. MMP-7 protein expression from SW1990 cell lysates was analyzed using Western blotting. β-actin served as an internal control. (B) SW1990 cells were starved in serum-free medium overnight and incubated with different concentrations of MSLN (0–2.0 μg/ml) for 12 h. MMP-7 mRNA and protein expression were then analyzed by qRT-PCR and Western blotting, respectively. Data from qRT-PCR represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to untreated control cells. (C, D) Scramble control or MUC16-KD SW1990 and Pa03C cells were serum-starved overnight and then incubated for 12 h in the presence or absence of MSLN (1 μg/ml). MMP-7 mRNA (C) and protein (D) levels were quantified by qRT-PCR and immunoblotting, respectively. Data represent the mean ± S.E. of at least three independent experiments. *, p < 0.01 and §, p < 0.05 with respect to the corresponding scramble control cells. (E) Conditioned media from scramble control or MUC16-KD pancreatic cancer cells pre-treated with MSLN (1 μg/ml) for 12 h were collected and analyzed for MMP-7 secretion. Data are reported as the fold of MMP-7 levels detected in the conditioned medium of each cell line compared to that in the medium of untreated scramble control cells. Data represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to corresponding untreated scramble control cells.

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Incubation, Quantitative RT-PCR, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: Scramble control or MUC16-KD SW1990 (A) or Pa03C (B) cells were incubated with MSLN (1 μg/ml) in the presence of either a neutralizing MMP-7 antibody (2 μg/ml) or an isotype control and then used in transwell invasion assays. Data are reported as normalized cell number that invaded through the transwell membrane relative to untreated scramble control (100%), and represent the mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001 (one-way ANOVA followed by Tukey test). (C) SW1990 and Pa03C pancreatic cancer cells were incubated with either human recombinant active MMP-7 (2 μg/ml) or vehicle control, and then used in transwell invasion assays. Data are reported as normalized cell number that invaded through the membrane relative to vehicle control-treated cells (100%), and represent the mean ± S.E. of three independent experiments. *, p < 0.05 with respect to vehicle control cells (Student's t-test).

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Incubation, Recombinant

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: Representative time-lapse images of scramble control and MUC16-KD SW1990 (A) and Pa03C (C) pancreatic cancer cells migrating through channels of prescribed dimensions ( W x H x L = 6 × 10 × 200 μm) using 10% FBS as chemoattractant. In all experiments, cells were incubated with or without MSLN (1 μg/ml) for 6 h before being seeded to the microchannel device. The average migration speed of individual scramble control and MUC16-KD SW1990 (B) and Pa03C (D) cells was determined over a 10 h period for > 30 cells from three independent experiments. *, p < 0.05 with respect to scramble control and MSLN-treated MUC16-KD cells. (E) The average migration speed of MSLN-stimulated SW1990 and Pa03C cells in the presence of either an MMP-7 neutralizing antibody (2 μg/ml) or an isotype control was quantified for > 30 cells from three independent experiments. *, p < 0.05 with respect to the control treatment. (F) The average migration speed of SW1990 and Pa03C cells in the presence or absence of human recombinant active MMP-7 (2 μg/ml) was quantified for > 30 cells from three independent experiments. *, p < 0.05 with respect to untreated control.

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Incubation, Migration, Recombinant

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: (A) Scramble control and MUC16-KD SW1990 cells were serum-starved overnight, and then treated with MSLN (1 μg/ml) for 12 h. The levels of phospho-ERK1/2 (Thr202/Tyr204) and phospho-p38 MAPK (Thr180/Tyr182) from whole cell lysates were analyzed by immunoblotting using specific Abs. Equal loading in each lane was ensured by the similar intensities of total ERK1/2, p38 MAPK, and β-actin. (B) SW1990 cells were incubated with MSLN (1 μg/ml) for 12 h in the presence or absence of the p38 MAPK inhibitor SB203580 (20 μM). The levels of phospho-p38 MAPK (Thr180/Tyr182) and MMP-7 expression from cell lysates were analyzed by Western blotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total p38 MAPK and β-actin. These Western blots are representative of three independent experiments, all revealing similar results. The intensity of bands was quantified using the NIH ImageJ software and then normalized with respect to the value obtained for the untreated control. (C, D) SW1990 cells were stimulated with MSLN (1 μg/ml) in the presence or absence of SB203580 (20 μM) or an MMP-7 antisense oligonucleotide (50 nM), and then subjected to either transwell invasion or microchannel motility assays. (C) Data are reported as percentage of untreated control cells that invaded through the transwell membrane, and represent the mean ± S.E. of three independent experiments. (D) The average migration speed of individual cells was determined over a 10 h period for > 30 cells from three independent experiments for each of the indicated conditions. *, p < 0.01.

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Incubation, Expressing, Software, Migration

Journal: Scientific Reports

Article Title: Mesothelin Binding to CA125/MUC16 Promotes Pancreatic Cancer Cell Motility and Invasion via MMP-7 Activation

doi: 10.1038/srep01870

Figure Lengend Snippet: The binding of MSLN to MUC16 present on the pancreatic cancer cell surface activates a p38 MAPK-dependent pathway, which in turn upregulates MMP-7 synthesis, resulting in increased invasive and migratory potentials. In the absence of MUC16 expression by pancreatic cancer cells, MSLN is capable of upregulating MMP-7 expression via activation of an ERK-dependent pathway.

Article Snippet: Western blotting assays were performed as previously described and stained with antibodies for MUC16, MSLN, MMP-7, MMP-9, β-actin, or signaling proteins including ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, p-Akt (Ser473), p38 MAPK, and p-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA).

Techniques: Binding Assay, Expressing, Activation Assay