Journal: Iranian Journal of Basic Medical Sciences

Article Title: Comparing the effect of plasma therapy with estradiol valerate in motor and cognitive behavioral disorders in ovariectomized old rats: Behavioral, biochemical, and protein expression

doi: 10.22038/ijbms.2024.81345.17608

Figure Lengend Snippet: Result of young plasma on the expression stages of p-CREB/CREB, BDNF, and SIRT1 protein expression levels in hippocampal tissue of ovariectomized old rats

Article Snippet: After obstructive, the primary antibodies, SIRT1, BDNF (108319, Abcam), phosphorylated and activated form of CREB (p-CREB), CREB, and GAPDH as housekeeping protein (sc-74504, SC- 7978, sc-377154, and sc-32233 respectively, Santa Cruz Biotechnology) were utilized.

Techniques: Clinical Proteomics, Expressing

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on cardiac muscle fibrosis and hypertrophy. ( A ) Picro-Sirius Red staining. Scale bar = 200 µm. ( B ) Quantification of Picro-Sirius Red staining. mRNA levels of ( C ) αSMA and ( D ) TGF-β1, two markers of fibrosis. ELISA assays were used to quantify ( E ) TGF-β and ( F ) the active phosphorylated form of SMAD2 (P¬SMAD2), a transcription factor mainly involved in TGF-β signalling. mRNA levels of ( G ) BNP and ( H ) ANP, two markers of hypertrophy. ( I ) ELISA assay was also used to quantify the levels of ANP. The percentage of stained areas was calculated in cardiac muscle sections, and the subsequent ratios are presented as relative expressions to WT values. mRNA levels were normalised to cyclophilin, and the subsequent ratios were presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on ApN receptors and signalling in the dystrophic cardiac muscle. mRNA levels of ( A ) AdipoR1 and ( B ) AdipoR2, adiponectin main receptors. ELISA assays were used to quantify ( C ) AdipoR1 and ( D ) AdipoR2. ( E ) The ratio of AdpoR1 over AdipoR2 mRNA levels was calculated within the cardiac muscle. ELISA assays were used to quantify ( F ) the active phosphorylated form of AMPKα (P-AMPK), ( G ) calcium/calmodulin-dependent protein kinase 2 (CAMKK2), and ( H ) peroxisome proliferator-activated receptor alpha (PPARα), ApN/AdipoRon, main signalling pathways in muscle. ( I ) mRNA levels of PGC-1α. ELISA assays were used to quantify ( J ) PGC-1α, ( K ) the active phosphorylated form of the p65 subunit of NF-κB (P-p65), a transcription factor mainly involved in inflammation, and ( L ) utrophin A (UTRN), a dystrophin analogue. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expression to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Antioxidants

Article Title: Striking Cardioprotective Effects of an Adiponectin Receptor Agonist in an Aged Mouse Model of Duchenne Muscular Dystrophy

doi: 10.3390/antiox13121551

Figure Lengend Snippet: Effects of AdipoRon treatment on cardiac muscle oxidative capacity, injury, and overall muscle function. mRNA levels of ( A ) ERRα and ( B ) mtTFA, two markers of mitochondrial biogenesis. ELISA assay was used to quantify ( C ) TOMM20, a marker of mitochondrial content. ( D ) Wire test where mice hanging time was recorded (s). ( E ) Fore-limb grip test and ( F ) fore- and hind-limb grip test, measuring muscle strength expressed in Gram-force relative to body weight (gf/gBW). ( G ) Treadmill running exercise, where the total distance covered on the third day was measured (m). ( H ) CK and ( I ) LDH plasma activities assessing muscle injury and expressed as IU/L. ( J ) ELISA assay was used to quantify the active phosphorylated form of RIP (P-RIP), an important regulator of cellular stress that triggers a regulated pathway for necrotic cell death called necroptosis. mRNA levels were normalised to cyclophilin, and the subsequent ratios are presented as relative expressions to WT values. Absorbance data are presented as relative expressions to WT values. Data are means ± SD; n = 6 mice per group for all ex vivo experiments. Data are means ± SD; n = 7–8 mice per group for all in vivo functional tests. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT mice. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs. mdx mice.

Article Snippet: ELISA assays were also used to quantify the levels of ANP, 4-Hydroxynonenal (HNE), TNFα, IL-1β, IL-10 (all from Abcam, Cambridge, UK), utrophin A (UTRN) (from Antibodies Online, Atlanta, GA, USA), TGF-β, AdipoR1, AdipoR2, the active phosphorylated form of Calcium/Calmodulin Dependent Protein Kinase Kinase 2 (CAMKK2), peroxisome proliferator-activated receptor α (PPARα), PPAR gamma coactivator 1 alpha (PGC-1α), Translocase of Outer Mitochondrial Membrane 20 (TOMM20), and the active phosphorylated form of Ribosome-inactivating protein (P-RIP) (all from MyBiosource—Bio-Connect Diagnostics B.V., Huissen, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Marker, Ex Vivo, In Vivo, Functional Assay

Journal: Children

Article Title: Sclerostin and Wnt Signaling in Idiopathic Juvenile Osteoporosis Using High-Resolution Confocal Microscopy for Three-Dimensional Analyses

doi: 10.3390/children11070820

Figure Lengend Snippet: Sclerostin expressing osteocytes in IJO bone biopsies. ( A ) Staining with an anti-sclerostin antibody (red) was assessed in healthy control (CTR) bone at 40×. ( A’ ) DAPI was used as a nuclear marker (blue). ( A” ) CTR bone staining of sclerostin and DAPI. ( B ) Staining with an anti-sclerostin antibody (red) was assessed in IJO. ( B’ ) DAPI was used as a nuclear marker (blue). ( B” ) IJO bone staining of sclerostin and DAPI. Arrows and arrowheads (insets) denote corresponding cells in red and blue channels with osteocytes expressing high levels of sclerostin. Trabecular (TB) bone and bone marrow (BM) are indicated. Scale bars are shown as 50 µm.

Article Snippet: For these analyses, primary antibodies included sclerostin and β-catenin, phosphorylated (phos) or unphosphorylated (active) forms (Cell Signaling; D13A1) were used, and secondary antibodies included AlexaFluor 568 (red) or AlexaFluor 488 (green).

Techniques: Expressing, Staining, Control, Marker

Journal: Children

Article Title: Sclerostin and Wnt Signaling in Idiopathic Juvenile Osteoporosis Using High-Resolution Confocal Microscopy for Three-Dimensional Analyses

doi: 10.3390/children11070820

Figure Lengend Snippet: High resolution three-dimensional sclerostin staining in IJO osteocytes. ( A ) Phase contrast imaging (DIC). ( B ) DIC overlaid with immunofluorescence staining with sclerostin antibody (red) of bone biopsy. ( C ) Heatmap of pixel intensity for sclerostin fluorescence intensity across z-stacks in IJO osteocytes (163×). ( D , E ) Insights of heatmap immunofluorescence staining of sclerostin. Scale bars are shown as 5 µm and 1 µm. Colors represent z-stack slice. ( F ) Quantification of sclerostin vesicle sizes.

Article Snippet: For these analyses, primary antibodies included sclerostin and β-catenin, phosphorylated (phos) or unphosphorylated (active) forms (Cell Signaling; D13A1) were used, and secondary antibodies included AlexaFluor 568 (red) or AlexaFluor 488 (green).

Techniques: Staining, Imaging, Immunofluorescence, Fluorescence

Journal: Children

Article Title: Sclerostin and Wnt Signaling in Idiopathic Juvenile Osteoporosis Using High-Resolution Confocal Microscopy for Three-Dimensional Analyses

doi: 10.3390/children11070820

Figure Lengend Snippet: Sclerostin-positive osteocytes colocalize with phosphorylated β-catenin. ( A ) Bone biopsies stained with a sclerostin antibody (red), ( A’ ) phosphorylated β-catenin (green), and ( A” ) DAPI (blue). ( A’’’ ) displays merged channels. Arrows indicate corresponding cells where sclerostin-positive osteocytes and phos β-catenin staining. Dotted white boxes are insets that highlight colocalization from the corresponding image in the row below. Magnified regions of ( B ) sclerostin, ( B’ ) phos β-catenin, and ( B” ) DAPI staining, in region marked from the row above. Trabecular (TB) bone and bone marrow (BM) are indicated. Scale bars are shown as 50 µm and 10 µm. ( B’’’ ) Merge of panels ( B – B” ).

Article Snippet: For these analyses, primary antibodies included sclerostin and β-catenin, phosphorylated (phos) or unphosphorylated (active) forms (Cell Signaling; D13A1) were used, and secondary antibodies included AlexaFluor 568 (red) or AlexaFluor 488 (green).

Techniques: Staining

Journal: Children

Article Title: Sclerostin and Wnt Signaling in Idiopathic Juvenile Osteoporosis Using High-Resolution Confocal Microscopy for Three-Dimensional Analyses

doi: 10.3390/children11070820

Figure Lengend Snippet: Sclerostin expressing osteocytes have low Wnt signaling activity. ( A ) IJO bone biopsies stained with a sclerostin antibody (red), ( A’ ) active β-catenin (green), and ( A” ) DAPI (blue). ( A’’’ ) displays the merged channels of ( A – A” ). Arrowheads indicate corresponding cells where sclerostin-positive osteocytes lack active β-catenin staining. ( B ) Bone biopsies stained with sclerostin, ( B’ ) phosphorylated (phos) β-catenin (green), and ( B” ) DAPI (blue). ( B’’’ ) displays merged channels of ( B – B” ). Arrows denote corresponding cells with colocalized sclerostin and phos β-catenin. Insets from dotted white boxes highlight colocalization. Trabecular (TB) bone and bone marrow (BM) are indicated. Scale bars are shown as 50 µm.

Article Snippet: For these analyses, primary antibodies included sclerostin and β-catenin, phosphorylated (phos) or unphosphorylated (active) forms (Cell Signaling; D13A1) were used, and secondary antibodies included AlexaFluor 568 (red) or AlexaFluor 488 (green).

Techniques: Expressing, Activity Assay, Staining

Journal: Cells

Article Title: Resveratrol Induces Myotube Development by Altering Circadian Metabolism via the SIRT1-AMPK-PP2A Axis

doi: 10.3390/cells13121069

Figure Lengend Snippet: Effect of resveratrol on metabolic factors. ( A ) SIRT1. ( B ) pAMPK/AMPK. ( C ) pACC/ACC. ( D ) pPP2A/PP2A. ( E ) Representative Western blots of all proteins at different glucose concentrations around the circadian cycle. C2C12 myotubes were incubated with resveratrol for 6 h and analyzed for an additional 24 h. Western blots were performed to determine protein levels. All time-points are expressed as average daily levels. Within each panel, protein levels display statistical significance ( p < 0.05) only when bars are presented with different letters.

Article Snippet: The blots were then incubated with antibodies against AMP-activated protein kinase (AMPK) and its phosphorylated form (pAMPK), protein phosphatase 2A (PP2A), pPP2A, SIRT1, acetyl CoA carboxylase (ACC), pACC, BMAL1, pBMAL1, AKT, P70S6K, pP70S6K, S6 and pS6 (Cell Signaling Technology, Beverly, MA, USA), ACTIN, pAKT, mTOR, pmTOR, MYOGENIN, CLOCK and CRY1 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA).

Techniques: Western Blot, Incubation

Journal: International journal of molecular sciences

Article Title: Water Extract of Angelica dahurica Inhibits Osteoclast Differentiation and Bone Loss.

doi: 10.3390/ijms241914715

Figure Lengend Snippet: Figure 3. Effects of WEAD on RANK signaling pathways in BMMs. (A) BMMs were treated with or without WEAD (200 µg/mL) and RANKL for the specified durations. The mRNA and protein levels of c-Fos and NFATc1 were assessed by real-time PCR and Western blotting, respectively. (B) BMMs were pre-treated with WEAD or vehicle for 3 h and then stimulated RANKL for the indicated time. Phosphorylated and non-phosphorylated forms of p38, JNK and IκBα were detected using Western blotting. (C) The mRNA expression of Irf8, MafB, Tm7sf4, Atp6v0d2 and CtsK. ** p < 0.01, *** p < 0.001 vs. vehicle control.

Article Snippet: Antibodies against phosphorylated forms including p-p38 (T180/Y182), p-JNK (T183/Y185), p-IκBα (S32) and their non-phosphorylated forms were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Protein-Protein interactions, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control