Journal: Scientific reports

Article Title: Disordered metabolism in mice lacking irisin.

doi: 10.1038/s41598-020-74588-7

Figure Lengend Snippet: Figure 6. Increased bone resorption in irisin lacking mice. (A, B) Sections of the distal shaft of the femur were stained with osteoprotegerin (OPG) (1:60) and receptor activator of nuclear factor-kB ligand (RANKL) (1:80) antibodies. Positive (black arrows) osteoblasts are shown, 200×. (C) Representative images of the distal metaphyseal region of the femur, together with cell counts (osteoclasts) per bone perimeter (Bpm). Red arrows, tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, 100×, (NIS-Elements Viewer, v4.2.0; https:// www.downza.cn/soft/2751-21.html); (D) serum levels of osteocalcin TRAP (with three replicates). (Graphpad Prism, v7.0, https://www.xue51.com/soft/3932.html). Data are presented as the mean ± SEM (n = 15 per group). *P < 0.05, **P < 0.01 compared to the WT group.

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit for osteocalcin (OCN) (E06917m), Alkaline Phosphatase (ALP) (E0200m), and Tartrate-resistant acid phosphatase (TRAP) (E08492m) were purchased from Elabscience (Wuhan, China).

Techniques: Staining

Journal: Heliyon

Article Title: Antidiabetic and hepatoprotection effect of butterfly pea flower ( Clitoria ternatea L.) through antioxidant, anti-inflammatory, lower LDH, ACP, AST, and ALT on diabetes mellitus and dyslipidemia rat

doi: 10.1016/j.heliyon.2024.e29812

Figure Lengend Snippet: Effect of CTE on liver ACP levels in DM and Dyslipidemia Rat Model. (a) ACP level (U/L); (b) ACP level (U/mg protein) * The data are presented as means±SD and were obtained from four repetitions. The experimental groups: Group I: Negative control (NC) comprising normal rats, Group II: Positive control (PC) consisting of rats with DM and dyslipidemia, Group III: PC+CTE 200 mg/kg BW (CTE200), Group IV: PC+CTE 400 mg/kg BW (CTE400), Group V: PC+CTE 800 mg/kg BW (CTE800), Group VI: PC+Simvastatin 0.9 mg/kg BW (SV), Group VII: PC+Glibenclamide 0.45 mg/kg BW (GC), and Group VIII: PC+Glibenclamide 0.45 mg/kg BW+Simvastatin 0.9 mg/kg BW (GS). Superscript signs on Figure 5a (a, b, c, cd, d, e) and Figure 5b (a, b, bc, c, d, e) indicate significant differences (p<0.05) among samples obtained from Tukey's HSD.

Article Snippet: The measurements included MDA levels (Elabscience, E-BC-K025-S), hepatic SOD levels (Elabscience, E-BC-K020), CAT levels (Elabscience, E-BC-K031), hepatic LDH levels (E-BC-K046-M), hepatic ACP levels (E-BC-K010-M), hepatic AST levels (Elabscience, E-BC-K236), hepatic ALT levels (Elabscience, E-BC-K235), hepatic IL-1β levels (E-EL-R0012), and hepatic CRP levels (Elabscience, E-EL-R0506) [ , ].

Techniques: Negative Control, Positive Control

Journal: Heliyon

Article Title: Antidiabetic and hepatoprotection effect of butterfly pea flower ( Clitoria ternatea L.) through antioxidant, anti-inflammatory, lower LDH, ACP, AST, and ALT on diabetes mellitus and dyslipidemia rat

doi: 10.1016/j.heliyon.2024.e29812

Figure Lengend Snippet: Proposed mechanism on how CTE influence metabolic syndrome rats' model * CTE is believed to have a hepatoprotective effect with phenolics and flavonoids content playing a crucial part in scavenging elevated free radicals. Also, flavonoids in CTE have shown the ability to increase the activity of antioxidant enzymes such as CAT and SOD. By elevating the activity of these enzymes, damage pancreatic β cells can be prevented, helping to maintain normal insulin levels in the body. CTE combats oxidative stress by countering free radicals, thereby protecting cells and tissues from damage. Consequently, levels of ACP and LDH, which indicate cell death and cellular injury, are reduced, suggesting decreased injury to the liver and an elevation in the level of liver protein. Additionally, the antioxidant anthocyanins and saponins present in CTE can further contribute to reducing the level of MDA, another indicator of oxidative stress. CTE also can suppress the pro-inflammatory cytokine IL−1β and reduce the levels of CRP, a protein that increases during inflammation, by inhibiting secondary enzymes.

Article Snippet: The measurements included MDA levels (Elabscience, E-BC-K025-S), hepatic SOD levels (Elabscience, E-BC-K020), CAT levels (Elabscience, E-BC-K031), hepatic LDH levels (E-BC-K046-M), hepatic ACP levels (E-BC-K010-M), hepatic AST levels (Elabscience, E-BC-K236), hepatic ALT levels (Elabscience, E-BC-K235), hepatic IL-1β levels (E-EL-R0012), and hepatic CRP levels (Elabscience, E-EL-R0506) [ , ].

Techniques: Activity Assay

Journal: Bone & Joint Research

Article Title: Melatonin alleviates senile osteoporosis by regulating autophagy and enhancing fracture healing in aged mice

doi: 10.1302/2046-3758.142.BJR-2024-0112.R2

Figure Lengend Snippet: Aged bone marrow-derived mesenchymal stem cells (BMMSCs) showed degenerative properties in osteogenesis and melatonin (MEL) promoted osteogenesis of aged BMMSCs. a) Haematoxylin and eosin (H&E) staining (40×) of proximal tibiae and b) micro-CT (μCT) imaging of distal femora of aged and young mice. c) Bone microstructure parameters such as bone mineral density (BMD), trabecular bone volume per total volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp). An independent-samples t -test was used to calculate the p-value in Figure 1c. d) Alkaline phosphatase (ALP) staining (20×) and e) Alizarin Red staining (20×) to analyze the osteogenesis of young and aged BMMSCs. f) Gene enrichment analysis of genes related to senile osteoporosis predicted using the Genecard database, DisGeNET database, and OMIM database, highlighting pathways where MEL metabolism and effects showed the highest enrichment in WikiPathways. Chi-squared test was used to calculate the p-value. g) MEL in serum was analyzed by enzyme-linked immunosorbent assay (ELISA). h) ALP staining (5×), i) ALP level analysis, j) Alizarin Red staining (5×), and k) mineralization level analysis were performed. *p < 0.05, significant differences between each indicated group analyzed using one-way analysis of variance (ANOVA). CTR, control group; IL-18, interleukin-18; MAPK, mitogen-activated protein kinase; ns, not significant; PI3K-AKT, phosphoinositide 3-kinase-protein kinase B; VEGFA-VEGFR2, vascular endothelial growth factor-vascular endothelial growth factor receptor 2.

Article Snippet: The concentration of tartrate-resistant acid phosphatase 5 (ACP5), OCN, MEL, in mouse serum were measured with Mouse Tartrate Resistant ACP5 ELISA Kit, Mouse Osteocalcin ELISA Kit, and Mouse Melatonin ELISA Kit (Elabscience Biotechnology, China), respectively, according to the manufacturer’s instructions.

Techniques: Derivative Assay, Staining, Micro-CT, Imaging, Enzyme-linked Immunosorbent Assay, Control

Journal: Bone & Joint Research

Article Title: Melatonin alleviates senile osteoporosis by regulating autophagy and enhancing fracture healing in aged mice

doi: 10.1302/2046-3758.142.BJR-2024-0112.R2

Figure Lengend Snippet: Inhibition of autophagy could counteract melatonin (MEL)-induced osteogenic promotion in aged mice. a) Micro-CT (μCT) imaging of distal femora and b) haematoxylin and eosin (H&E) staining (40×) of proximal tibiae of aged mice treated with MEL for six weeks (50 mg kg -1 body weight per day). c) Bone microstructure parameters such as bone mineral density (BMD), trabecular bone volume per total volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp) were measured by μCT scanning. d) to f) Immunohistopathology (200×) showed the osteogenic-related protein presentations of d) matricellular molecular osteocalcin (OCN) and e) osteogenic marker Osterix in proximal tibia bone tissue, and f) integral optical density (IOD) was calculated. g) to i) Concentrations of g) bone resorption marker ACP5, h) osteogenic marker OCN, and i) MEL in serum were analyzed by enzyme-linked immunosorbent assay (ELISA). *p < 0.05, significant differences between each indicated group analyzed using Fisher’s exact test, one-way analysis of variance (ANOVA), or Tukey’s post-hoc test. CTR, control group; ns, not significant.

Article Snippet: The concentration of tartrate-resistant acid phosphatase 5 (ACP5), OCN, MEL, in mouse serum were measured with Mouse Tartrate Resistant ACP5 ELISA Kit, Mouse Osteocalcin ELISA Kit, and Mouse Melatonin ELISA Kit (Elabscience Biotechnology, China), respectively, according to the manufacturer’s instructions.

Techniques: Inhibition, Micro-CT, Imaging, Staining, Marker, Enzyme-linked Immunosorbent Assay, Control

Journal: Bone & Joint Research

Article Title: Melatonin alleviates senile osteoporosis by regulating autophagy and enhancing fracture healing in aged mice

doi: 10.1302/2046-3758.142.BJR-2024-0112.R2

Figure Lengend Snippet: Administration of melatonin (MEL) has therapeutic effects on bone fracture healing in aged mice. a) Representative radiograph and micro-CT (μCT) of femora in aged mice (n = 6 per group) four weeks after open femoral mid-shaft fracture. b) Nonunion frequency and c) μCT measurement of bone volume per total volume (BV/TV) and bone mineral density (BMD) in callus area of the fractured femora four weeks after open femoral mid-shaft fracture. d) Haematoxylin and eosin (H&E) staining (50×) of fractured femur. e) Ratio of newly formed bone, cartilage, and fibrous tissue area and quantification of the area of new bone formation. f) Immunohistopathology (200×) of fractured femur, showing the osteogenic-related protein matricellular protein osteocalcin (OCN) in the fracture callus. The integral optical density (IOD) of OCN was calculated. g) Osteoblast counts in the fracture callus were assessed by counting the number of osteoblasts on each H&E-stained section, with osteoblast number/bone perimeter (N.Ob/B.Pm) (/mm) determined using Image-Pro Plus software (Media Cybernetics, USA). Blue arrows indicate typical osteoblasts in the sections, highlighting their distribution in the fracture callus. h) Evaluation of osteoclasts in fracture callus. The numbers of osteoclasts were counted based on tartrate-resistant acid phosphatase (TRAP)-stained sections, and osteoblast number/total area (N.OC/T.Ar) (/mm 2 ) was determined by Image-Pro Plus software. i) Protein presentations from bone marrow-derived mesenchymal stem cells (BMMSCs) extracted from tibiae after fracture healing of autophagy markers LC3BII, Beclin, and the osteogenesis marker OCN. *p < 0.05, significant differences between each indicated group analyzed using Fisher's exact test, one-way analysis of variance (ANOVA), or Tukey’s post-hoc test. CTR, control group; GelMA, methacrylated gelatin; ns, not significant.

Article Snippet: The concentration of tartrate-resistant acid phosphatase 5 (ACP5), OCN, MEL, in mouse serum were measured with Mouse Tartrate Resistant ACP5 ELISA Kit, Mouse Osteocalcin ELISA Kit, and Mouse Melatonin ELISA Kit (Elabscience Biotechnology, China), respectively, according to the manufacturer’s instructions.

Techniques: Micro-CT, Staining, Software, Derivative Assay, Marker, Control

Journal: Cell Reports

Article Title: Defining a Spinal Microcircuit that Gates Myelinated Afferent Input: Implications for Tactile Allodynia

doi: 10.1016/j.celrep.2019.06.040

Figure Lengend Snippet:

Article Snippet: Chicken anti-Prostatic acid phosphatase (1:1000) , Aves Labs. Inc., OR, USA. , Cat#PAP; RRID: AB_2313557.

Techniques: Virus, Plasmid Preparation, Software

Journal: Science advances

Article Title: CD8 + T cell targeting of tumor antigens presented by HLA-E.

doi: 10.1126/sciadv.adm7515

Figure Lengend Snippet: Fig. 6. Stimulation of MHC-E–restricted, PAP-specific CD8+ T cells by PAP-expressing K562 cells. (A) Immunoblot of cell lysates of indicated K562-derived cell lines. Expres- sion of endogenous and transfected HLA-E in K562 cells was demonstrated with two HLA-E–specific antibodies MEM-E/02 and 3D12 (5, 90, 91). Note that nontransfected K562 cells express low amounts of HLA-E (88). V5 epitope–tagged RhPAP was detected with PAP-specific (MAB6240, R&D Systems) or V5-specific mouse monoclonal antibodies. A GAPDH-specific mAb was used as loading control. (B) ICS for IFNγ and TNFα of CD8+ T cells from three 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cells in the presence or absence of VL9 peptide. (C) Results from five independent experiments showing the average relative frequency (background-subtracted and normalized to K562-E/PAP) of CD69 and IFNγ and/or TNFα-positive CD8+ T cells from 68-1 RhCMV/RhPAP-immunized RM responding to the indicated cell lines in the presence or absence of VL9. Individual results are shown in data file S3. Statistical significance was determined by paired t test (not significant, P > 0.05). (D) Immunoblot for HLA-E and FLAG-tagged acid phosphatases. Lysates of stable cell lines of AA7 cells (= K562 cells deleted for HLA-E; fig. S7) transfected with HLA-E alone or together with human PAP, ACP2, or ACPT were electrophoretically separated and probed with the indicated antibodies by immunoblot. (E) ICS for IFNγ and TNFα of CD8+ T cells from three 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cell lines. (F) Average frequencies (+SEM) of CD69 and IFNγ and/or TNFα-positive CD8+ T cells responding to the indicated cell lines were background subtracted and normalized to AA7-E/PAP. Individual results are shown in data file S3. Statistical significance was determined using paired t test.

Article Snippet: Following blocking with 5% milk in PBS–0.1% Tween- 20 (Thermo Fisher Scientific) (PBS- T), membranes were probed with the following antibodies in 5% milk in PBS- T: anti–MHC- E 3D12 (1:1000; Invitrogen 14- 9953- 82), anti–MHC- E MEM- E/02 (1:5000; Invitrogen MA1- 19304), antiFLAG epitope (1:5000; Sigma- Aldrich #F3165), anti- V5 epitope (1:500; Invitrogen #37- 7500), anti–glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Invitrogen #MA5- 15738), anti- HA epitope (1:2000; clone HA- 7, Sigma- Aldrich H9658), anti- PAP (1:500; MAB6240, R&D Systems) anti–MHC- I heavy chain (1:5000; HC10, Thermo Fisher Scientific), or anti- RhCMV IE2 (15 μg/ml; clone IIA5.2) (53).

Techniques: Expressing, Western Blot, Derivative Assay, Transfection, Bioprocessing, Control, Stable Transfection

Journal: Science advances

Article Title: CD8 + T cell targeting of tumor antigens presented by HLA-E.

doi: 10.1126/sciadv.adm7515

Figure Lengend Snippet: Fig. 7. Recognition of PCa cell lines by MHC-E–restricted, PAP-specific CD8+ T cells. (A) Immunoblot of cell lysates of the indicated cell lines for HLA-E, PAP, HLA-Ia, and GAPDH. Antibodies used were MEM-E/02 for HLA-E (90), HC10 for HLA-Ia heavy chains (92), and MA5-15738 (Invitrogen) for GAPDH. The anti-PAP antibody (MAB6240 R&D Systems) detected a lower molecular weight band in DU145 cells that is likely nonspecific since DU145 cells are known to be PAP negative (93, 94). (B) ICS for IFNγ and TNFα production of CD8+ T cells from a 68-1 RhCMV/RhPAP-immunized RM after coincubation with the indicated cell lines in the presence or absence of VL9 peptide. In addition, we inhibited potential MHC-II presentation by adding anti-DR and CLIP peptide to all stimulations. (C) Average frequencies (+SEM) of CD69 and IFNγ and/or TNFα-positive CD8+ T cells from 68-1 RhCMV/RhPAP-immunized RM after background subtraction. The number of repeat experiments is indicated. Individual results are shown in data file S3. Statistical significance was calculated using paired Mann-Whitney U test (not significant, P > 0.05).

Article Snippet: Following blocking with 5% milk in PBS–0.1% Tween- 20 (Thermo Fisher Scientific) (PBS- T), membranes were probed with the following antibodies in 5% milk in PBS- T: anti–MHC- E 3D12 (1:1000; Invitrogen 14- 9953- 82), anti–MHC- E MEM- E/02 (1:5000; Invitrogen MA1- 19304), antiFLAG epitope (1:5000; Sigma- Aldrich #F3165), anti- V5 epitope (1:500; Invitrogen #37- 7500), anti–glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Invitrogen #MA5- 15738), anti- HA epitope (1:2000; clone HA- 7, Sigma- Aldrich H9658), anti- PAP (1:500; MAB6240, R&D Systems) anti–MHC- I heavy chain (1:5000; HC10, Thermo Fisher Scientific), or anti- RhCMV IE2 (15 μg/ml; clone IIA5.2) (53).

Techniques: Western Blot, Molecular Weight, MANN-WHITNEY

Journal: Science advances

Article Title: CD8 + T cell targeting of tumor antigens presented by HLA-E.

doi: 10.1126/sciadv.adm7515

Figure Lengend Snippet: Fig. 8. Recognition of primary PCa cells by MHC-E–restricted, PAP-specific CD8+ T cells. Left: ICS for IFNγ and TNFα production by CD8+ T cells from two 68-1 RhCMV/ RhPAP-immunized RM after coincubation with PCa cell suspensions in the presence or absence of VL9 peptide. In addition, we inhibited MHC-II presentation by adding anti-DR antibody and CLIP peptide in all stimulations. Right: Summary of results from the indicated number of primary PCa samples showing the average frequency of CD69 and IFNγ and/or TNFα-positive CD8+ T cells from 68-1 RhCMV/RhPAP-immunized RM in the absence or presence of VL9 peptide after background subtraction. Indi- vidual results are shown in data file S3. Statistical significance was calculated using paired t test.

Article Snippet: Following blocking with 5% milk in PBS–0.1% Tween- 20 (Thermo Fisher Scientific) (PBS- T), membranes were probed with the following antibodies in 5% milk in PBS- T: anti–MHC- E 3D12 (1:1000; Invitrogen 14- 9953- 82), anti–MHC- E MEM- E/02 (1:5000; Invitrogen MA1- 19304), antiFLAG epitope (1:5000; Sigma- Aldrich #F3165), anti- V5 epitope (1:500; Invitrogen #37- 7500), anti–glyceraldehyde phosphate dehydrogenase (GAPDH; 1:5000; Invitrogen #MA5- 15738), anti- HA epitope (1:2000; clone HA- 7, Sigma- Aldrich H9658), anti- PAP (1:500; MAB6240, R&D Systems) anti–MHC- I heavy chain (1:5000; HC10, Thermo Fisher Scientific), or anti- RhCMV IE2 (15 μg/ml; clone IIA5.2) (53).

Techniques: