Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 1. Vitamin D3 increases legumain expression, activity, and secretion in pre-osteoblastic cells. (A) The nucleotide sequence of the LGMN gene promoter region with annotations of potential vitamin D-responsive elements (VDRE; red) relative to the transcription start site (TSS). (B–F) Human BMSC- TERT cells (20,000 cells/cm2) were incubated with 1,25(OH)2D3 (B–F; 10, 50 or 100 nM), 25(OH)D3 (C–F; 100, 250, 500 or 1000 nM) or an equal volume of ethanol (control, 0 nM) in osteoblast induction medium for seven days before harvesting. (B) Legumain mRNA expression relative to housekeeping control (GAPDH) (2−∆∆CT; n = 3). (C) One representative immunoblot of legumain (proform 56 kDa, mature form 36 kDa) and GAPDH (housekeeping) in cell lysates (n = 3). (D) Quantification of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in C (n = 3). (E) Legumain activity (dF/s) in cell lysates adjusted for the total protein concentration (µg/mL) (n = 6–9). (F) Secreted legumain (pg/mL) in conditioned media measured by ELISA and adjusted for the total protein concentration in the corresponding cell lysates (n = 3–5). (B,D–F) Data represent mean ± SEM. (B,D) Kruskal–Wallis test. (E,F) One-way ANOVA. * p < 0.05 vs. 0 nM 1,25(OH)2D3 or 25(OH)D3. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Sequencing, Incubation, Control, Western Blot, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 2. Treatment with 25(OH)D3 increases legumain levels and activity in wild-type mice. Wild-type mice (Lgmn+/+) were treated with 50 µg/kg 25(OH)D3 (n = 7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (A) Legumain mRNA expression relative to the geometric mean of CT values of four housekeeping controls in kidney, liver, and spleen (2−∆∆CT; n = 5). (B) One representative immunoblot of legumain and GAPDH in kidney, liver, and spleen (n = 3). (C) Quantifi- cation of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH (housekeeping) in kidney, liver, and spleen from immunoblots represented in (C) (n = 3). (D) Legumain activity (dF/s) in kidney, liver, and spleen adjusted for total protein concentration (µg/mL, n = 5). (E) Legumain plasma concentration (ng/mL) measured by ELISA (n = 5). (F) Cor- relation between legumain (ng/mL and 1,25(OH)2D3 (pmol/L) concentrations in plasma (n = 5). (A,C,E) Two-tailed unpaired Student’s t-test. (D) Mann–Whitney test. Data represent mean ± SEM. * p < 0.05. (F) Simple linear regression. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Activity Assay, Control, Injection, Expressing, Western Blot, Protein Concentration, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 3. Legumain is required for VDBP processing and regulation. (A) Purified VDBP from human plasma (1.9 µM) was incubated in legumain assay buffer (pH 5.8) at 37 ◦C with or without purified active bovine legumain (2 µM) for 5 h before gel electrophoresis and immunoblotting of VDBP (n = 1). (B–H) Wild-type (Lgmn+/+) and legumain-deficient (Lgmn−/−) mice were treated with 50 µg/kg 25(OH)D3 (n = 6–7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (B) One representative immunoblot of VDBP and GAPDH (housekeeping) in kidney and liver (n = 4). (C–F) Quantification of VDBP immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in (B) (n = 4). (C) Hepatic VDBP 45 kDa immunoband. (D) Renal VDBP 45 kDa immunoband. (E) Hepatic VDBP 55 kDa immunoband. (F) Renal VDBP 55 kDa immunoband. (G) Plasma VDBP concentration (µg/mL) was measured by ELISA (n = 6–7). (H) Hepatic VDBP mRNA expression relative to the geometric mean of CT values of four house- keeping controls (2−∆∆CT, n = 5). (C–H) Data represent mean ± SEM. Two-way ANOVA. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. different genotype, same treatment. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Purification, Clinical Proteomics, Incubation, Nucleic Acid Electrophoresis, Western Blot, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 5. Graphical representation of the suggested interplay between vitamin D and legumain. Left panel: Vitamin D (VD3) promotes legumain expression and activity through transcriptional upregulation of the legumain gene (LGMN). The free fraction of circulating VD3 metabolites diffuse through plasma membranes. 25-hydroxyvitamin D (25(OH)D3) is hydroxylated by 1α-hydroxylase (CYP27B1), forming the active metabolite 1α,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 binds to the nuclear vitamin D receptor (VDR) and promotes transcription of legumain (LGMN). Synthesized prolegumain is either sorted and activated in the endolysosomal system or released to the extracellular environment. Right panel: In the proximal tubular epithelium, 25(OH)D3 bound to vitamin D binding protein (VDBP) is internalized from the tubular lumen through a megalin/cubilin- mediated process. The vitamin D metabolite is released, enabling subsequent hydroxylation by 1α-hydroxylase (CYP27B1) or 24-hydroxylase (CYP24A1), and VDBP is cleaved by legumain in the endolysosomal system. VDBP cleavage by legumain might be important in controlling the systemic level of vitamin D metabolites. Created with BioRender.com (accessed on 11 December 2023).

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Synthesized, Binding Assay

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Expression of TIMP-3 in vitreous fluid samples. Equal volumes (15 μl) of vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) ( n = 12) and non-diabetic patients with rhegmatogenous retinal detachment (RD) ( n = 12) were subjected to gel electrophoresis and the presence of TIMP-3 was detected by Western blot analysis. A representative set of samples is shown.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) attenuates retinal inflammation and prevents diabetes-induced blood-retinal barrier (BRB) breakdown. The BRB breakdown [panel (A) ] was quantified with the fluorescein isothiocyanate-conjugated dextran technique after treatment with intravitreal injection of 350 μM TIMP-3 in 5 μl in one eye and the same volume of phosphate-buffered saline (PBS) in the contralateral eye of rats 10 weeks after induction of diabetes. Results are expressed as mean ± standard deviation of five rats in each group. Evaluation of inflammatory mediators was evaluated shortly after diabetes induction (see section “Materials and Methods”). Western blot analysis of rat retinas was performed to evaluate protein expression levels of phospho-ERK1/2 [panel (B) ], the p65 subunit of NF-κB [panel (C) ], intercellular adhesion molecule-1 (ICAM-1) [panel (D) ], and vascular endothelial growth factor (VEGF) [panel (E) ]. Results are expressed as mean ± standard deviation of 12 rats in each group. One-way ANOVA and independent t -test were used for comparisons between the three and two groups, respectively, panels (A–E) . * p < 0.05 compared with non-diabetic controls. # p < 0.05 compared with PBS-treated diabetic rats.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Injection, Standard Deviation, Western Blot, Expressing

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were left untreated or treated with high-glucose (HG, 25 mM) [panel (A) ] cobalt chloride (CoCl 2 ) (300 μM) [panel (B) ] or tumor necrosis factor -α (TNF-α) (50 ng/ml) [panel (C) ] for 24 h or TIMP-3 (100 ng/ml) for 1 h followed by HG, CoCl 2 , or TNF-α. For HG treatment, cultures containing 25 mM mannitol were used as a control. Levels of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were quantified in the culture media by ELISA. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparison between three groups and two groups, respectively. * p < 0.05 compared with values obtained from control cells. # p < 0.05 compared with values obtained from cells treated with HG, CoCl 2 , or TNF-α.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before increasing the sugar content of the cultures [25 mM of mannitol as control or 25 mM of glucose (HG)]. After 24 h levels of the p65 subunit of NF-κB [panel (A) ], phospho-ERK1/2 [panel (B) ], caspase-3 [panel (C) ], and ADAM17 [panel (D) ] in cell lysates was determined by Western blot analysis. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from cells treated with mannitol. # p < 0.05 compared with values obtained from cells treated with HG.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Incubation, Western Blot, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: THP-1 monocytes were left untreated or treated with TIMP-3 (100 ng/ml) for 24 h. Subsequently, the THP-1 cells were fluorescently labeled and adhesion to a human retinal microvascular endothelial cell (HRMEC) monolayer was assessed [panel (A) ]. Results are expressed as median (interquartile range) from two independent experiments (each treatment condition: 6 wells) (* p < 0.05; Mann-Whitney test). Alternatively, HRMECs were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium, tumor necrosis factor-α (TNF-α) (25 ng/ml) [panel (B) ] or vascular endothelial growth factor (VEGF) (50 ng/ml) [panel (C) ] for 24 h. Adhesion of fluorescently labeled monocytic cells to the HRMEC monolayer was assessed. Results are expressed as median (interquartile range) from three independent experiments (each treatment condition: 6 wells). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively (RFU = relative fluorescence unit). HRMECs were left untreated or were stimulated with TNF-α (50 ng/ml) for 24 h with/without a 1-h pre-incubation with TIMP-3 (100 ng/ml). Protein expression of vascular cell adhesion molecule-1 (VCAM-1) [panel (D) ] and intercellular adhesion molecule-1 (ICAM-1) [panel (E) ] was determined by Western blot analysis. Results are expressed as mean ± standard deviation from three independent experiments (each treatment condition: 8 wells). One-way ANOVA and independent t -test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from untreated cells. # p < 0.05 compared with values obtained from cells treated with TNF-α or VEGF.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Labeling, MANN-WHITNEY, Incubation, Fluorescence, Expressing, Western Blot, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) inhibits vascular endothelial growth factor (VEGF)-mediated human retinal microvascular endothelial cell (HRMEC) migration, chemotaxis and proliferation. A scratch was made in confluent monolayers of overnight starved HRMECs with a micropipette tip. Subsequently, the cultures were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium or vascular endothelial growth factor (VEGF) (50 ng/ml) for 24 h. Cells were visualized using an inverted microscope. Three independent experiments were performed. Each experiment was done in triplicate and 6–8 independent field images were taken for the migration analysis which was done by using image J software. In the figure one representative image is shown [panel (A) ]. Results are expressed as median (interquartile range). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with untreated cells. # p < 0.05 compared with VEGF-treated cells. Chemotaxis and proliferation of HRMECs stimulated with 10 ng/ml VEGF was modulated by a 30-min pre-incubation with TIMP-3 (10 or 100 ng/ml). Cell migration was monitored using the xCELLigence RTCA-DP system. The median (interquartile range) percentage of inhibition of VEGF-induced chemotaxis [panel (B) ] or proliferation [panel (C) ] is shown. In total five chemotaxis experiments were performed and conditions were tested in duplicate or triplicate within 1 experiment. For proliferation, 6 experiments were performed and conditions were tested at least in triplicate within 1 experiment. * p < 0.05; Mann-Whitney test (compared with VEGF).

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Migration, Chemotaxis Assay, Incubation, Inverted Microscopy, Software, MANN-WHITNEY, Inhibition

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Structure prediction of TMEM95 protein (left) using IZUMO1 (right) as template, created by SWISS-MODEL software. ( B ) Tmem95 KO allele generated following CRISPR-mediated edition. CRISPR target sequence and PAM are depicted in blue and purple letters, respectively. ( C ) The deletion of 10 bp altered Tmem95 ORF. Large letters indicate the aminoacid sequence corresponding to the codons (DNA sequence) shown in smaller letters below. ( D ) Western Blot images for TMEM95, IZUMO1 and β-tubulin proteins from protein extracts from WT or KO sperm. Graph on right indicates the abundance of IZUMO1 in WT and KO extracts. ( E ) Immunocytochemistry images of KO and WT sperm stained with an antibody against TMEM95 and the acrosomal stain PNA. TMEM95 localized to the acrosomal cap in acrosome intact sperm and in the equatorial segment after acrosome reaction. ( F ) Immunocytochemistry images of acrosome intact (upper images) or reacted (lower images) WT sperm stained against IZUMO1 and TMEM95. Both proteins relocalize to the equatorial segment following acrosome reaction.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Software, Generated, CRISPR, Sequencing, Western Blot, Immunocytochemistry, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Immunoblotting of protein extracts obtained from WT and KO epididymal sperm samples following two lysis protocols. In lysis protocol #1, sperm were re-suspended in 4X reducing SDS Sample Buffer and boiled for 10 min. In lysis protocol #2, sperm were re-suspended in 1% Octyl β-D-glucopyranoside solution in PBS and incubated on ice for 30 min . Supernatants were probed with anti-TMEM95 (MyBiosource MBS7004333), anti-IZUMO1 (Abcam ab211623) or anti-β-TUBULIN (Sigma T8328) antibodies. ( B ) Gel electrophoresis of PCR products amplified from cDNA obtained from testis, seminal vesicle (S.V.) prostate (prost.), epididymis (epid.) or a negative control testis RNA not retrotranscribed (RT-) to detect Gapdh and Tmem95 transcripts. ( C ) Immunoblotting of protein extracts obtained from WT epididymal sperm, testis or accessory glands (seminal vesicle and prostate). Same antibodies than ( A ). ( D ) Uncropped images of the WB used to generate . ( E ) WB images used for the quantification of IZUMO1 and β-TUBULIN shown in . Four samples were used for quantification (marked with asterisk), as β-TUBULIN band on samples #4 was dispersed, leading to inaccurate quantification.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Western Blot, Lysis, Incubation, Nucleic Acid Electrophoresis, Amplification, Negative Control

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Immunocytochemistry images of WT sperm stained with PNA and IZUMO1 antibody (upper row) or PNA and TMEM95 antibody (lower row) in the absence of permeabilizing agents. Despite the absence of permeabilizing agents (30 min fixation in 4% PFA without Triton X-100), TMEM95 and inner acrosomal (PNA) and acrosomal membrane (IZUMO1) markers were detected. ( B ) IZUMO1 relocates to the equatorial segment following acrosome reaction in TMEM95 KO sperm. Upper row shows one acrosome intact spermatozoon. Lower rows show WT and KO acrosome reacted sperm where IZUMO1 has relocated to the equatorial segment.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Immunocytochemistry, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Binding analysis using the AVEXIS assays shows that the soluble recombinant TMEM95 ectodomain does not interact with JUNO nor with IZUMO1. The entire ectodomains of the named proteins were expressed in HEK293-6E cells either as biotinylated baits or as pentameric beta-lactamase-tagged preys. Bait proteins were immobilised on streptavidin-coated plates and captured prey proteins quantified by measuring the absorbance of a colorimetric reaction product of the beta-lactamase substrate, nitrocefin. The CD200R (bait)-CD200 (prey) binding pair was used as positive control. The same prey, CD200R, was tested against TMEM95 and is shown as negative control. Bars represent means + s.d.; n = 3. ( B ) HEK293 cells stably expressing the N-terminal half of GFP (GFP1-7) and mouse JUNO stained with a highly avid IZUMO1 probe. ( C ) HEK293 cells stably expressing the C-terminal half of GFP (GFP8-11) and mouse IZUMO1 stained with a highly avid JUNO. ( D ) TMEM95 does not induce fusion when expressed in HEK293T cells in the presence of JUNO and IZUMO1 using a GFP-complementation cell fusion assay. HEK293T cells expressing either half of GFP and either JUNO or IZUMO1 were mixed and their fusogenic ability visualized by GFP fluorescence. The IZUMOI-expressing cells were either mock transfected prior to mixing (Control), transfected with Syncitin a , as a positive fusion control, or Tmem95 . By contrast to the cells transfected with Syncytin a , Tmem95 did not induce cell fusion. Cell nuclei are stained with DAPI and scale bar represents 20 µm.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Binding Assay, Recombinant, Positive Control, Negative Control, Stable Transfection, Expressing, Staining, Cell Fusion Assay, Fluorescence, Transfection

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet: ( A ) Amino acid sequence of FKBP1A underlined in blue fused via a short linker to the N-terminal region of GFP (GFP 1-7 ) (green underline). ( B ) Amino acid sequence of the C-terminal region of GFP (GFP 8-11 ) highlighted in green, fused to RB, underlined in blue. (C, C´, D and D´) The cells expressing the GFP1-7 stably express mouse Juno and the cells expressing the GFP8-11 stably express mouse Izumo1. To establish functional cell surface expression of both Juno and Izumo1 on the cells, noth cell lines were stained with the corresponding pentameric FLAG-tagged Izumo1 and Juno preys. The cells expressing mouse Juno were stained with the Izumo1 prey but not Juno, and the cells expressing mouse Izumo1 stained with the Juno prey but not Izumo1. Images are representative of at least three independent replicates. Nuclei are stained with DAPI (blue) and scale bars represents 10 µm.

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Sequencing, Expressing, Stable Transfection, Functional Assay, Staining

Journal: eLife

Article Title: TMEM95 is a sperm membrane protein essential for mammalian fertilization

doi: 10.7554/eLife.53913

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit Anti-IZUMO1 , AbCam , Ab211623; RRID: AB_2650506 , .

Techniques: Generated, CRISPR, Recombinant, Plasmid Preparation, Sequencing