Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Myeloid-specific Pfkfb3 deficiency attenuates LPS-induced inflammatory responses. (A) Representative images (left) and quantification (right) for immunohistochemical staining of the neutrophil marker Ly6G in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (B) Representative images (left) and quantification (right) for immunohistochemical staining of the macrophage marker Mac2 in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (C–E) qPCR analysis of the mRNA levels of Il1b (C) , Il6 (D) and Nos2 (E) in the lung of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 6). (F,G) ELISA analysis of Il1b (F) and Il6 (G) in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 4). (H) NO levels in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 3–4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for Pfkfb3 WT vs. Pfkfb3 ΔMϕ (unpaired two-tailed Student's t test).

Article Snippet: After antigen retrieval with Antigen Unmasking Solution (H-3301, Vector Laboratories, Burlingame, CA, USA) at 98°C for 10 min, sections were blocked with avidin solution with 10% normal rabbit serum for 1 h at room temperature, and incubated in biotin blocking solution with primary antibodies against Mac2 (3 μg/mL, CL8942F, Cedarlane, Burlington, NC, USA), or Ly6G (3 μg/mL, 551459, BD biosciences, San Jose, CA, USA) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Marker, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The FASEB Journal

Article Title: Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

doi: 10.1096/fj.202400446R

Figure Lengend Snippet: Experimental design. (A) Schema depicting experimental plan for testing pro‐tumorigenicity of protein products of MSLN gene. Red bar shows MPF protein, which is expected to be secreted. Blue bar shows MSLN protein. Lighter blue indicates residues C‐terminal to the GPI anchor which is required for membrane‐bound MSLN. Constructs missing the GPI‐anchor are expected to be secreted‐only. Darker blue residues indicate those which may contribute to sMSLN. Y318A point mutation ablates MSLN interaction with MUC‐16. (B) Schematic showing each MSLN variant complemented into KO cells.

Article Snippet: The following primary and secondary antibodies were used for MSLN: Mouse‐human MSLN (BioXCell) and Goat Anti‐Mouse IgG (H+L)‐HRP Conjugate (Bio‐Rad, #1706516); for GAPDH: GAPDH (Cell Signaling, Cat#:5147) and Goat Anti‐Rabbit IgG (H+L)‐HRP Conjugate (Bio‐Rad; #1706515).

Techniques: Membrane, Construct, Mutagenesis, Variant Assay

Journal: The FASEB Journal

Article Title: Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

doi: 10.1096/fj.202400446R

Figure Lengend Snippet: Assessing the activity of MPF versus membrane‐bound mature MSLN. KLM1 MSLN KO cells were stably transfected with empty vector (KO+vec), MSLNf, or MPFf. (A) KLM1 derivative cells were assessed for membrane‐bound MSLN expression by flow cytometry. (B) Growth rate of the cell lines on tissue culture plastic was measured. There was no significant difference between the groups. (C–F) Cell lines were injected IP into nude mice and allowed to grow for ~6 weeks. (C) Total burden of peritoneal tumor was measured, *** p < .001. (D, E) MSLN and MPF concentration in tumor lysate was measured by ELISA assay. (F) Tumor burden of co‐injected +MSLNf and +MPFf cells was assessed, * p < .05; ns, not significant.

Article Snippet: The following primary and secondary antibodies were used for MSLN: Mouse‐human MSLN (BioXCell) and Goat Anti‐Mouse IgG (H+L)‐HRP Conjugate (Bio‐Rad, #1706516); for GAPDH: GAPDH (Cell Signaling, Cat#:5147) and Goat Anti‐Rabbit IgG (H+L)‐HRP Conjugate (Bio‐Rad; #1706515).

Techniques: Activity Assay, Membrane, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

doi: 10.1096/fj.202400446R

Figure Lengend Snippet: Assessing the activity of secreted‐only MSLN in KO cells. (A) Schema depicting truncation mutant (Δ591) to remove the GPI anchoring site and prevent membrane association of mature MSLN. KLM1 MSLN KO cells were stably transduced with WT and Y318A (Mu) Δ591 expression vectors and single cell clones were isolated. (B) Immunoblot of Δ591 clones to assess for MSLN expression. (C) Conditioned medium of Δ591 clones plated at equal density was assayed for MSLN concentration by ELISA. (D) Representative experiment showing growth rate of the KO+ Δ591 cell lines on tissue culture plastic as measured by counting cell number in triplicate wells. (E) Cells were suspended in soft agar and colonies were counted after ~3 weeks. Figure depicts raw data of triplicate wells (marker) in each experiment (bar). ** p < .01 or *** p < .001 indicate statistically significant difference as compared to parent for at least 2 of 3 experiments (in same direction of change). (F, G) Cell lines were injected IP into nude mice and allowed to grow for ~6 weeks. (F) Tumors were lysed and MSLN expression was assayed by immunoblot. (G) Total burden of peritoneal tumor dissected from the mouse abdominal cavity, * p < .05. Each point represents one animal. Results were confirmed by repeat using other Δ591WT and Δ591Mu clones. (H) MUC‐16 surface expression was assessed by flow cytometry. Left— Representative tracing. Right— Summary of geometric means in relation to Parent over multiple experiments.

Article Snippet: The following primary and secondary antibodies were used for MSLN: Mouse‐human MSLN (BioXCell) and Goat Anti‐Mouse IgG (H+L)‐HRP Conjugate (Bio‐Rad, #1706516); for GAPDH: GAPDH (Cell Signaling, Cat#:5147) and Goat Anti‐Rabbit IgG (H+L)‐HRP Conjugate (Bio‐Rad; #1706515).

Techniques: Activity Assay, Mutagenesis, Membrane, Stable Transfection, Transduction, Expressing, Clone Assay, Isolation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Marker, Injection, Flow Cytometry

Journal: The FASEB Journal

Article Title: Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

doi: 10.1096/fj.202400446R

Figure Lengend Snippet: Overexpression of sMSLN inhibits the pro‐tumorigenic activity of cells expressing membrane‐bound MSLN. (A) Proposed model showing how sMSLN might block cell–cell association by interfering with interactions between MUC‐16 and membrane‐bound MSLN. (B–H) KLM1 cells were stably transduced with Δ591 MSLN expression vectors and pooled cells were used for experiments. (B) Expression of MSLN in conditioned medium as detected by ELISA. (C) Representative experiment showing growth rate of the cell lines on tissue culture plastic as measured by counting cell number in triplicate wells for each time point. No significant difference in growth was observed. (D–F) Cell lines were injected IP into nude mice and allowed to grow for ~6 weeks, * p < .05; *** p < .001; **** p < .0001; ns, not significant. (D) Total burden of peritoneal tumor dissected from the mouse abdominal cavity. Each point represents one animal. (E) Tumors were lysed and MSLN expression was assayed by immunoblot. Each lane is lysate from one mouse tumor. (F) Serum MSLN expression was assayed by ELISA. (G, H) Indicated cells were tagged with CellTracker dye and visualized by fluorescence microscopy. Representative images of multiple replicates with four to eight fields imaged per replicate. (G) Labeled cells were plated at equal density onto low adherence plates for 24 h before visualization. (H) Labeled cells were injected IP into nude mice. Mice were euthanized 4 h later and peritoneal lavage fluid was collected for visualization.

Article Snippet: The following primary and secondary antibodies were used for MSLN: Mouse‐human MSLN (BioXCell) and Goat Anti‐Mouse IgG (H+L)‐HRP Conjugate (Bio‐Rad, #1706516); for GAPDH: GAPDH (Cell Signaling, Cat#:5147) and Goat Anti‐Rabbit IgG (H+L)‐HRP Conjugate (Bio‐Rad; #1706515).

Techniques: Over Expression, Activity Assay, Expressing, Membrane, Blocking Assay, Stable Transfection, Transduction, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, Fluorescence, Microscopy, Labeling

Journal: The FASEB Journal

Article Title: Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

doi: 10.1096/fj.202400446R

Figure Lengend Snippet: Excess of sMSLN prevents cell clustering. (A) Schema showing method used to produce MSLN‐containing CM. Mock cells underwent transient transfection with Cas9 vector like MSLN KO cells, but no gRNA was included, so they retain endogenous MSLN expression (MSLN+). Transduction of KO cells with full‐length MSLN WT or Y318A expression construct produces the KO+WT and KO+Mu cell lines which overexpress MSLN (MSLN++). (B) ELISA to assess MSLN concentration of CM from equal numbers of cells plated for each type. (C) Cells were tagged with CellTracker dye then plated at equal density onto low adherence plates for 24 h before visualization by fluorescence microscopy. Representative images of multiple replicates with 4–8 fields imaged per replicate. (D) Schema for treatment of KLM1 and T3M4 KO cells with CM prior to RNA harvest. (E) PCA plots following RNA deep‐sequencing of CM‐treated KO cells. (F) Quantitation and directionality of GSEA pathway changes in KO cells treated with KO CM (negative control) as compared to MSLN‐containing CMs.

Article Snippet: The following primary and secondary antibodies were used for MSLN: Mouse‐human MSLN (BioXCell) and Goat Anti‐Mouse IgG (H+L)‐HRP Conjugate (Bio‐Rad, #1706516); for GAPDH: GAPDH (Cell Signaling, Cat#:5147) and Goat Anti‐Rabbit IgG (H+L)‐HRP Conjugate (Bio‐Rad; #1706515).

Techniques: Transfection, Plasmid Preparation, Expressing, Transduction, Construct, Enzyme-linked Immunosorbent Assay, Concentration Assay, Fluorescence, Microscopy, Sequencing, Quantitation Assay, Negative Control

Journal: The FASEB Journal

Article Title: Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

doi: 10.1096/fj.202400446R

Figure Lengend Snippet: Soluble MSLN exposure results in release of IL‐1α. (A, B) Venn diagrams outlining GSEA pathway changes that occur when MSLN KO cells are exposed to sMSLN‐containing CM as compared with exposure to CM from MSLN KO cells. Pathways with statistically significant differential expression are listed. Those in bold were significantly changed in both KLM1 and T3M4. (C, D) KO cells were treated with stock CM isolated from KO, Mock, +WT or +Mu cells for 0 or 4 h, ** p < .01; **** p < .0001; ns, not significant. The concentration of IL‐1α (C) or LIF (D) in CM was then measured by ELISA.

Article Snippet: The following primary and secondary antibodies were used for MSLN: Mouse‐human MSLN (BioXCell) and Goat Anti‐Mouse IgG (H+L)‐HRP Conjugate (Bio‐Rad, #1706516); for GAPDH: GAPDH (Cell Signaling, Cat#:5147) and Goat Anti‐Rabbit IgG (H+L)‐HRP Conjugate (Bio‐Rad; #1706515).

Techniques: Quantitative Proteomics, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of galectin-3 and apoptosis in placental villi from patients with missed abortion during early pregnancy

doi: 10.3892/etm.2019.7227

Figure Lengend Snippet: Galectin-3 mRNA expression in chorionic villi of the SA and MA groups. The relative mRNA expression levels of galectin-3 were determined by RT-qPCR in chorionic villi tissue samples from patients in the SA and MA group. (A) The expression level of galectin-3 was significantly increased in the MA group compared with the SA group. (B) The expression of galectin-3 in early MA group (<4 weeks) is decreased compared with the SA group. The expression level of galectin-3 in the MA group gradually increases with time. *P<0.05 vs. SA group. SA, spontaneous abortion; MA, missed abortion; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; 4 w, 4 weeks.

Article Snippet: The samples were incubated with the primary antibody anti-galectin 3 (1:200; cat. no. PB9081; Boster Biotechnology, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of galectin-3 and apoptosis in placental villi from patients with missed abortion during early pregnancy

doi: 10.3892/etm.2019.7227

Figure Lengend Snippet: Galectin-3 protein expression in chorionic villi of the SA and MA groups. The relative mRNA expression levels of galectin-3 were determined by ELISA in chorionic villi tissue samples from patients in the SA and MA group. (A) The galectin-3 protein expression level is significantly higher in the MA group compared with the SA group. (B) The galectin-3 protein expression level in the early MA group (<4 weeks) is not significantly different compared with the SA group; whilst the galectin-3 protein expression level in the late MA group (>4 weeks) was significantly higher compared with the SA group. *P<0.05 vs. SA group. SA, spontaneous abortion; MA, missed abortion; 4 w, 4 weeks.

Article Snippet: The samples were incubated with the primary antibody anti-galectin 3 (1:200; cat. no. PB9081; Boster Biotechnology, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of galectin-3 and apoptosis in placental villi from patients with missed abortion during early pregnancy

doi: 10.3892/etm.2019.7227

Figure Lengend Snippet: Galectin-3 protein expression in SA and MA groups determined by IHC. Galectin-3 protein expression was examined in chorionic villi tissue samples from patients in the (A) negative control, (B) SA group and (C) MA group (magnification, ×400). Red arrows indicate galectin-3-positive expression in cells stained by DAB and blue arrows indicate cell nuclei counterstained with hematoxylin. SA, spontaneous abortion; MA, missed abortion; IHC, immunohistochemistry; DAB, diaminobenzidine.

Article Snippet: The samples were incubated with the primary antibody anti-galectin 3 (1:200; cat. no. PB9081; Boster Biotechnology, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Negative Control, Staining, Immunohistochemistry

Journal: Experimental and Therapeutic Medicine

Article Title: Expression of galectin-3 and apoptosis in placental villi from patients with missed abortion during early pregnancy

doi: 10.3892/etm.2019.7227

Figure Lengend Snippet: Quantification of galectin-3 protein expression in SA and MA groups determined by IHC. SA, spontaneous abortion; MA, missed abortion; IHC, immunohistochemistry.

Article Snippet: The samples were incubated with the primary antibody anti-galectin 3 (1:200; cat. no. PB9081; Boster Biotechnology, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Immunohistochemistry

Journal: Cancers

Article Title: Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients

doi: 10.3390/cancers14010144

Figure Lengend Snippet: Antibodies used for mass cytometry.

Article Snippet: 3153026B , Fluidigm , galectin-3 (Gal-3) , 153 Eu.

Techniques: Cytometry