Journal: Marine Drugs

Article Title: A Novel Aldisine Derivative Exhibits Potential Antitumor Effects by Targeting JAK/STAT3 Signaling

doi: 10.3390/md21040218

Figure Lengend Snippet: 11c inhibited constitutive and IL6-induced activation of STAT3. ( A , B ) Constitutive STAT3 activation cells DU145 ( A ) and A549 ( B ) were treated with 11c at the specific concentration for 2 h. ( C , D ) Hela ( C ) and MB231 ( D ) cells were pretreated with 11c at the indicated concentrations for 2 h before treatment with IL-6 (20 ng/mL) for 10 min. Whole cell lysates were processed for Western blot and probed with anti-p-STAT3 (Tyr 705) antibody. The relative expressions compared to the loading control protein β-Actin were measured by ImageJ from 3 individual experiments and shown as bar graphs on the right side of each Western blot. Error bars indicate means ± SD. p < 0.05 (*), significant, Student’s t -test, one-way ANOVA.

Article Snippet: Obtained recombinant human IL6 (Cat. 216-16) was from PeproTech.

Techniques: Activation Assay, Concentration Assay, Western Blot, Control

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: Biomedicines

Article Title: Exploring the Role of the TAS2R16 Protein and Its Single Nucleotide Variants in Pituitary Adenoma Development

doi: 10.3390/biomedicines12092022

Figure Lengend Snippet: Distributions of TAS2R16 rs860170, rs978739, and rs1357949 genotypes, and alleles in patients with PA and those in control group by PA invasiveness.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) was performed for this study, and the Abbexa Human Taste Receptor Type 2 Member 16 (TAS2R16) ELISA kit (Abbexa LTD; Cambridge, UK) was used.

Techniques: Control

Journal: Biomedicines

Article Title: Exploring the Role of the TAS2R16 Protein and Its Single Nucleotide Variants in Pituitary Adenoma Development

doi: 10.3390/biomedicines12092022

Figure Lengend Snippet: Binomial logistic regression analysis of TAS2R16 rs860170, rs978739, and rs1357949 in non-invasive PA group and control group.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) was performed for this study, and the Abbexa Human Taste Receptor Type 2 Member 16 (TAS2R16) ELISA kit (Abbexa LTD; Cambridge, UK) was used.

Techniques: Control

Journal: Biomedicines

Article Title: Exploring the Role of the TAS2R16 Protein and Its Single Nucleotide Variants in Pituitary Adenoma Development

doi: 10.3390/biomedicines12092022

Figure Lengend Snippet: TAS2R16 serum levels in PA and control groups.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) was performed for this study, and the Abbexa Human Taste Receptor Type 2 Member 16 (TAS2R16) ELISA kit (Abbexa LTD; Cambridge, UK) was used.

Techniques: Control

Journal: Biomedicines

Article Title: Exploring the Role of the TAS2R16 Protein and Its Single Nucleotide Variants in Pituitary Adenoma Development

doi: 10.3390/biomedicines12092022

Figure Lengend Snippet: Distribution of TAS2R16 rs860170, rs978739, and rs1357949 genotypes in PA and control groups according to TAS2R16 serum concentration.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) was performed for this study, and the Abbexa Human Taste Receptor Type 2 Member 16 (TAS2R16) ELISA kit (Abbexa LTD; Cambridge, UK) was used.

Techniques: Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: The histone code reader Spin1 controls skeletal muscle development

doi: 10.1038/cddis.2017.468

Figure Lengend Snippet: Surviving Spin1 M5 mice exhibit major defects in soleus, tibialis anterior, and diaphragm. ( a,b ) Appearance ( a ) and average body weight ( b ) of Spin1 M5 and control mice ( n =5 females in each category) at 30 weeks of age. Error bars represent +S.D., ** P <0.01. ( c ) Degeneration of the soleus (SOL) muscle in adult Spin1 M5 compared with control mice exemplified at 16 weeks of age. ( d ) Hind limb muscles (gastrocnemius (GC), plantaris (PL), soleus, tibialis anterior (TA), extensor digitorum longus (EDL), and quadriceps (QC)) of Spin1 M5 and control mice at 30 weeks of age. Arrows point at the soleus embedded in gastrocnemius and plantaris, which is visible in control but degenerated in Spin1 M5 mice. ( e ) Hematoxylin & eosin (H&E) staining of gastrocnemius, soleus, TA, and EDL muscle of Spin1 M5 and control mice at 30 weeks of age. ( f ) Fiber types in glycolytic (white) or oxidative (red) parts of the TA of Spin1 M5 and control mice at 15 weeks of age (top and middle rows) observed by immunofluorescence (IF) staining. Tissue sections were stained with selective antibody directed against MHC-I (purple), MHC-IIb (cyan), MHC-IIa (red), and MHC-IIx (green). For comparison, NADH staining was included (bottom row). Fibers with abnormal NADH staining are marked with arrows. Corresponding fibers in each column of images are squared

Article Snippet: For immunofluorescence staining the following primary antibodies were used: SPIN1(5865) 1 μ g/ml; Pax7 (PAX7, DSHB, batch 7/2/15) 2 μ g/ml; Tcf4 (6H5-3, Millipore, Darmstadt, Germany, 05-511, batch 2155406) 10 μ g/ml; Ankrd1 (Proteintech Group, Manchester, UK, 11427-1-AP, batch 1951) 1:100; Ankrd2 (Proteintech Group, 11821-1-AP, batch 7649) 1:100; dystrophin (Abcam, Cambridge, UK, ab15277, batch GR226781-6) 1:500; MHC-I (NOQ7.5.4D, Sigma, Munich, Germany, M8421, batch 035M4792V) 1:2000; MHC-IIa (SC-71, DSHB, batch 4/7/16) 1:10; MHC-IIx (6H1, DSHB, batch 3/3/16) 1:6; MHC-IIb (BF-F3, DSHB, batch 5/12/16) 1:20; MCH, skeletal, fast (MY-32, Sigma, M4276, batch 083M4790V) 1:1000; GNZ (anti-GFP, Abcam, ab13970, batch GR236651) 1:1000; normal rabbit IgG (Santa Cruz, Heidelberg, Germany, sc-2027).

Techniques: Control, Muscles, Staining, Immunofluorescence, Comparison