Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway

doi: 10.3390/ijms24043807

Figure Lengend Snippet: Representative images and quantitative analysis for Akt, FoxOs and eNOS expressions in STZ-induced diabetic mice with or without febuxostat treatment. ( a ) Representative immunoblot showing Akt, FoxOs and eNOS expression levels in mouse kidneys. ( b ) Quantitative analyses for phosphor-Ser 473 Akt/total Akt, ( c ) Quantitative analyses for phospho-Ser 256 FoxO1/total-FoxO1. ( d ) Quantitative analyses for phospho-Ser 253 FoxO3a/total-FoxO3a. ( e ) Quantitative analyses for phospho-Ser 1173 eNOS/total eNOS, * p < 0.05 and *** p < 0.001 vs. Cont, †† p < 0.01 and ††† p < 0.001 vs. Feb and ‡ p < 0.05, ‡‡ p < 0.01, and ‡‡‡ p < 0.001 vs. STZ group.

Article Snippet: The membrane was incubated with primary antibodies against the following target proteins: VEGFR1 (Boster, Pleasanton, CA, USA), VEGFR2 (St John’s Laboratory, London, UK), VEGFR3 (NSJ Bioreagent Logo, San Diego, CA, USA), NOX1 and NOX4 (Lifespan Biosciences, Seattle, WA, USA), NOX2 (BD Biosciences, San Jose, CA, USA), total Akt, phospho-Ser 473 Akt, SOD1 and SOD2 (Cell Signaling Technology, Danvers, MA, USA), total FoxO1, phospho-Ser 256 FoxO1, total FoxO3a, and phospho-Ser 253 FoxO3a (Novus Biologicals, Centennial, CO, USA), total endothelial nitric oxide synthase (eNOS, EMD Millipore, Middlesex County, MA, USA), and phospho-Ser 1173 eNOS (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway

doi: 10.3390/ijms24043807

Figure Lengend Snippet: Representative images and quantitative analysis for NOX1, NOX2, NOX4, FoxO3a, and eNOS according to VEGFR1 or VEGFR3 inhibition in HG-treated human GECs with or without febuxostat. ( a ) Representative immunoblot images of NOX1, NOX2, NOX4, FoxO3a and eNOS. ( b ) Quantitative analyses of NOX1/GAPDH. ( c ) Quantitative analyses of NOX2/GAPDH. ( d ) Quantitative analyses of NOX4/GAPDH. ( e ) Quantitative analyses for phospho-Ser 253 FoxO3a/total FoxO3a. ( f ) Quantitative analyses for phospho-Ser 1173 eNOS/total eNOS. * p < 0.05, ** p < 0.01, and *** p < 0.001, Fit1: anti-Flt1 peptide, SAR: SAR131675.

Article Snippet: The membrane was incubated with primary antibodies against the following target proteins: VEGFR1 (Boster, Pleasanton, CA, USA), VEGFR2 (St John’s Laboratory, London, UK), VEGFR3 (NSJ Bioreagent Logo, San Diego, CA, USA), NOX1 and NOX4 (Lifespan Biosciences, Seattle, WA, USA), NOX2 (BD Biosciences, San Jose, CA, USA), total Akt, phospho-Ser 473 Akt, SOD1 and SOD2 (Cell Signaling Technology, Danvers, MA, USA), total FoxO1, phospho-Ser 256 FoxO1, total FoxO3a, and phospho-Ser 253 FoxO3a (Novus Biologicals, Centennial, CO, USA), total endothelial nitric oxide synthase (eNOS, EMD Millipore, Middlesex County, MA, USA), and phospho-Ser 1173 eNOS (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Inhibition, Western Blot

Journal: Journal of ethnopharmacology

Article Title: Gastrodin alleviates cisplatin nephrotoxicity by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway.

doi: 10.1016/j.jep.2023.117282

Figure Lengend Snippet: Fig. 3. GAS regulates SIRT1 expression and decreases acetylated levels of FOXO3A in CP-induced AKI mice. Representative immunoblot images were obtained using the kidney cortex harvested 3 days after CP injection. Quantitative analyses for SIRT1 (A), SIRT6 (B), FOXO3A (C), and ac-FOXO3A (D). n = 3 mice per group. Scale bar: 50 μm **P < 0.01 compared to control mice. #P < 0.05, ##P < 0.01 compared to CP-treated mice. ac = acetylated; CP = cisplatin; FOXO3A = forkhead box O3A; GAS = gastrodin; SIRT1 = sirtuin-1.

Article Snippet: Immunoprecipitation was performed overnight at 4 ◦C with 4.2 μg of primary anti-FOXO3A antibody (Cat: NBP2-16521) (Novus), or IgG control antibody (Cat:3900S) (CST).

Techniques: Expressing, Western Blot, Injection, Control

Journal: Journal of ethnopharmacology

Article Title: Gastrodin alleviates cisplatin nephrotoxicity by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway.

doi: 10.1016/j.jep.2023.117282

Figure Lengend Snippet: Fig. 4. GAS regulates key FOXO3A-affected molecules and alleviates lipid peroxidation in CP-induced AKI mice. Representative immunoblot images. The kidney cortex used for Western blotting was harvested 3 days after the injection of CP. Quantitative analyses for GPX4 (A), MnSOD (B), catalase (C), and 4-HNE (D). n = 3–4 mice per group. Tissue iron (E), MDA (F), and GSH (G) were detected. n = 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated mice; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to CP-injured mice without GAS treatment. AKI = acute kidney injury; CP = cisplatin; GAS = gastrodin; GPX4 = glutathione peroxidase 4; GSH = glutathione; MnSOD = Mn-containing superoxide dismutase; MDA = malondialdehyde; ns = no statistically significant difference; 4-HNE = 4-hydroxynonenal.

Article Snippet: Immunoprecipitation was performed overnight at 4 ◦C with 4.2 μg of primary anti-FOXO3A antibody (Cat: NBP2-16521) (Novus), or IgG control antibody (Cat:3900S) (CST).

Techniques: Western Blot, Injection

Journal: Journal of ethnopharmacology

Article Title: Gastrodin alleviates cisplatin nephrotoxicity by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway.

doi: 10.1016/j.jep.2023.117282

Figure Lengend Snippet: Fig. 6. FOXO3A transcriptionally regulates GPX4 expression. (A) HK-2 cells were transfected with siControl or siFOXO3A for 48 h, and then cell lysates were subjected to Western blotting. n = 3 biological replicates. ***P < 0.001 compared to siControl HK-2 cells. (B) HK-2 cells were stably transfected with empty vectors or FOXO3A overexpression lentivirus and then treated with or without CP. GPX4 expression was detected after 24 h. Results were representative of n = 3–4 biological replicates. *P < 0.05 compared to untreated empty vector stably transfected cells. #P < 0.05 compared to CP-treated empty vector stably transfected cells. (C) Predicted DNA-binding element (DBE) of FOXO3A in the human GPX4 gene promoter region using JASPAR online analyses (http://jaspar.genereg.net). (D) ChIP- qPCR analysis of FOXO3A binding to the GPX4 promoter. Protein-bound chromatin was isolated from HK-2 cells and immunoprecipitated with FOXO3A antibodies. n = 3 biological replicates. **P < 0.01 compared to IgG-ChIP. (E) Dual-luciferase reporter assays were performed to measure the transcriptional activity of GPX4 in HK-2 cells overexpressing FOXO3A. Luciferase reporter plasmid containing GPX4 promoter sequence (or blank sequence) were transfected into the Control (empty vector) or FOXO3A-overexpressing HK-2 cells. Results represent the average of n = 3 biological replicates. **P < 0.01 compared to empty vector cells transfected with a luciferase reporter plasmid containing GPX4 promoter sequence and Renilla luciferase plasmid. ChIP = chromatin immunoprecipitation; CP = cisplatin; FOXO3A = forkhead box O3A; GPX4 = glutathione peroxidase 4; HK-2 = the immortalized human renal tubular epithelial cell line.

Article Snippet: Immunoprecipitation was performed overnight at 4 ◦C with 4.2 μg of primary anti-FOXO3A antibody (Cat: NBP2-16521) (Novus), or IgG control antibody (Cat:3900S) (CST).

Techniques: Expressing, Transfection, Western Blot, Stable Transfection, Over Expression, Plasmid Preparation, Binding Assay, ChIP-qPCR, Isolation, Immunoprecipitation, Luciferase, Activity Assay, Sequencing, Control, Chromatin Immunoprecipitation

Journal: Journal of ethnopharmacology

Article Title: Gastrodin alleviates cisplatin nephrotoxicity by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway.

doi: 10.1016/j.jep.2023.117282

Figure Lengend Snippet: Fig. 7. SIRT1 regulates GPX4 expression through the deacetylation of FOXO3A in CP-induced HK-2 cells. (A and B) HK-2 cells were transfected with siControl or siSIRT1 for 48 h, and then cell lysates were subjected to Western blotting. n = 3 biological replicates. *P < 0.05 compared to siCON cells. (C to F) HK-2 cells were stably transfected with empty or SIRT1 overexpressed lentivirus. After CP treatment of cells for 24 h, protein expression of SIRT1, GPX4, FOXO3A, and ac-FOXO3A was detected. n = 3 biological replicates. *P < 0.05, ***P < 0.001 compared to empty vector stably transfected cells. #P < 0.05, ###P < 0.001 compared to CP- treated cells stably transfected with empty vector. CP = cisplatin; FOXO3A = forkhead box O3A; GPX4 = glutathione peroxidase 4; HK-2 = the immortalized human renal tubular epithelial cell line; SIRT1 = sirtuin-1.

Article Snippet: Immunoprecipitation was performed overnight at 4 ◦C with 4.2 μg of primary anti-FOXO3A antibody (Cat: NBP2-16521) (Novus), or IgG control antibody (Cat:3900S) (CST).

Techniques: Expressing, Transfection, Western Blot, Stable Transfection, Plasmid Preparation

Journal: Journal of ethnopharmacology

Article Title: Gastrodin alleviates cisplatin nephrotoxicity by inhibiting ferroptosis via the SIRT1/FOXO3A/GPX4 signaling pathway.

doi: 10.1016/j.jep.2023.117282

Figure Lengend Snippet: Fig. 9. Overexpression of FOXO3A or SIRT1 alleviates CP-induced cell injury in HK-2 cells. (A and B) Cell membrane injury was evaluated by PI staining (original magnification × 100). PI cannot penetrate the normal cell membrane but can penetrate the injured membrane and stain the nucleus red. Scale bar: 100 μm. n = 3 biological replicates. ###P < 0.001 compared to stably transfected empty vector cells. CP = cisplatin; FOXO3A = forkhead box O3A; HK-2 = the immortalized human renal tubular epithelial cell line; PI = propidium iodide; SIRT1 = sirtuin-1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Immunoprecipitation was performed overnight at 4 ◦C with 4.2 μg of primary anti-FOXO3A antibody (Cat: NBP2-16521) (Novus), or IgG control antibody (Cat:3900S) (CST).

Techniques: Over Expression, Membrane, Staining, Stable Transfection, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: ( A ) Endogenous proteins in total lysates of MCF-7 cells were subjected to IP with an antibody (Ab) as indicated followed by immunoblotting (IB) with an anti-βTrCP1 or anti-FOXO3 Ab. A rabbit IgG or an isotype mouse IgG were included as an IP negative control. The input IB data indicated the integrity of lysates used for IP. ( B ) 293T cells were cotransfected with HA-ubiquitin (Ub) and FOXO3-Myc or FOXO3-S644A expression vectors plus IKKβ or a control vector (pCDNA3.1) as indicated. At 24 hours after transfection, cells were treated with either DMSO (control) or the IKKβ inhibitor (2 µM) plus the proteasome inhibitor MG-132 (10 µM) for 5 hours. Total lysates were prepared and sonicated extensively for preventing non-covalent protein-protein associations, and subjected to IP with an anti-myc Ab and followed by IB analysis with an anti-HA Ab. The typical pattern of HA-tagged Ub was highlighted. The input IB data were performed with Abs against FOXO3, IKKβ, phospho-IκBα (p-IκBα), IκBα, βTrCP1, and β-actin to show the expressions of transfected vectors in cells as indicated, the effect of the IKKβ inhibitor (as indicated by the repression of p-IκBα level), and the integrity of each protein in total lysates used for IP. ( C ) IP was performed with the lysates of 293T cells that were cotransfected with the expression vectors as indicated, treated with MG-132 as described above, and followed by IB with an anti-HA Ab. The input IB data were performed with Abs against myc-tag, FOXO3, βTrCP1, IKKβ, and β-actin to show the expressions of transfected vectors in cells and the integrity of lysates used for IP. ( D ) Firstly, 293T cells were transfected with either control-shRNA or βTrCP1-shRNA vectors. Twenty four hours post transfection, these cells were cotransfected with the expression vectors as indicated. Then, at 24 hours after cotransfection, cells were treated with MG-132 (20 µM) for 4 hours. The total lysates of these 293T cells were prepared and IP was performed as described above and followed by IB with an anti-HA Ab. The input IB data were performed with Abs against myc-tag, βTrCP1, IKKβ, and β-actin as described. ( E ) 293T cells were cotransfected with various doses of βTrCP1 expression vector and mutant βTrCP1ΔF vector and other expression vectors as indicated. IP was performed with the lysates of these transfected 293T cells, treated with MG-132 as described above, and followed by IB with an anti-HA Ab. The input IB data were performed with Abs against FOXO3, IKKβ, βTrCP1, and β-actin to show the expressions of transfected vectors and endogenous proteins in transfected cells and the integrity of lysates used for IP. ( F ) 293T cells were cotransfected with various doses of IKKβ expression vector and other expression vectors as indicated. IP was performed with the lysates of these transfected 293T cells, treated with MG-132, and followed by IB with an anti-HA Ab as described above. The input IB data were performed with the indicated Abs as described above. ( G ) In vitro ubiquitination assays. The indicated target proteins GST-FO(1–300), GST-FO(301–673), and GST (negative control) were incubated with or without E3-ligase protein GST-βTrCP1 in Ub buffer containing E1, E2 (UbcH5b), Mg-ATP, biotinylated ubiquitin, and IKKβ and analyzed by SDS-PAGE and IB with streptavidin conjugated with HRP or an anti-GST Ab as protein controls (lower panel). ( H ) The indicated target proteins were incubated with wild-type E3-ligase protein GST-βTrCP1 or mutant GST-βTrCP1ΔF in Ub buffer, and analyzed as described above. The positive signals are highlighted with *.

Article Snippet: A specific anti-FOXO3 Ab was obtained from Imgenex Corp. Abs against β-tubulin, β-actin, hemagglutinin (HA), myc-tag and Flag-tag were purchased from Sigma.

Techniques: Western Blot, Negative Control, Ubiquitin Proteomics, Expressing, Control, Plasmid Preparation, Transfection, Sonication, shRNA, Cotransfection, Mutagenesis, In Vitro, Incubation, SDS Page

Journal: PLoS ONE

Article Title: Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: ( A ) The stability of FOXO3 protein was assayed by cycloheximide (CHX) chase in 293T cells co-transfected with FOXO3-myc and βTrCP1 or a control vector (pCDNA3.1). Lysates of the transfected cells were prepared at 2, 4, 6, 8 hour (h) and 0 h (control) after addition of CHX, and subjected to immunoblotting (IB) with an anti-myc Ab. Graph shows results of densitometric analysis of 3 CHX chase experiments (mean ± SD) using 0 h (control) as 100%. ( B ) MCF-7 cells were transfected with small interfering RNA (siRNA) targeting βTrCP1 or control-siRNA (control). Total lysates of the transfected cells were subjected to IB analysis with an Ab against βTrCP1 or FOXO3 or β-actin (loading control) as indicated. ( C ) Total lysates of wild-type (Wt) and βTrCP1(–/–) MEF cells were subjected to IB analysis with an Ab against FOXO3 or βTrCP1 or β-actin (loading control). ( D ) βTrCP1(–/–) and βTrCP1(+/+) MEF cells were fixed, and the expression and subcellular localization of endogenous FOXO3 was detected using an anti-FOXO3 Ab and followed by an Alexa Fluor 546 (red) -conjugated secondary Ab, and analyzed with fluorescence microscopy. A fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) was used to visualize the nuclei. An average of ∼200 cells stained with anti-FOXO3 Ab were analyzed and a histogram shows the relative FOXO3 expression level in MEF cells. ( E and F ) Total lysates of 293T cells cotransfected with FRE-luc (firefly luciferase (luc) reporter containing FOXO-responsive elements), pRL-TK (renilla luc as a transfection control for normalization), βTrCP1, FOXO3 (A) or FOXO3-S644A (FO-S644A) (B), plus IKKβ or Akt-DD (an active Akt) as indicated and subjected to luc assays.

Article Snippet: A specific anti-FOXO3 Ab was obtained from Imgenex Corp. Abs against β-tubulin, β-actin, hemagglutinin (HA), myc-tag and Flag-tag were purchased from Sigma.

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Small Interfering RNA, Expressing, Fluorescence, Microscopy, Staining, Luciferase

Journal: PLoS ONE

Article Title: Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: ( A ) GST-pull down in vitro assays. Whole lysates from 293T cells overexpression of IKKβ were incubated with the GST-FOXO3 [GST-FO (1–300) and GST-FO (301–673)] fusion proteins as indicated and GST alone (negative control), and analyzed by SDS-PAGE and immunoblotting (IB) with an anti-βTrCP1 Ab (upper panel) and an anti-GST Ab as protein controls (lower panel). ( B ) The same lysates were incubated with the eight GST-FO sequential fusion fragments spanning the entire carboxy (C)-terminal region of FOXO3 (301–673) as indicated and GST, and analyzed as described. ( C ) In vitro kinase assays. Lysates of 293T transfected with HA-IKKβ were IP with an anti-HA, and kinase assays were performed with fusion proteins GST-FO (579–625) and GST-FO (579–625)-AA, whose Ser-586 and Ser-590 residues were mutated to Ala (A) residues, and GST protein (negative control). ( D ) Lysates from 293T cells were incubated with fusion proteins GST-FO (301–673) and GST-FO (301–673)AAA, whose Ser-586, Ser-590, and Ser-644 residues were mutated to Ala residues in the C-terminal domain of FOXO3, and GST protein, and analyzed by IB with an anti-βTrCP1 Ab (upper panel) and an anti-GST Ab (lower panel). The significant signal is indicated with *. ( E ) MCF-7 cells were treated with DMSO (control vehicle for the IKKβ inhibitor shown in F below) or untreated, fixed, and the subcellular localizations and co-localization of endogenous FOXO3 and βTrCP1 proteins were detected using an anti-FOXO3 and a specific anti-βTrCP1 Ab (obtained from K. Strebel at NIH) and followed by an Alexa Fluor 594- or 488-conjugated secondary Ab, respectively, and fluorescence microscopy. DAPI was used to show the nuclei, and co-localization of FOXO3 with βTrCP1 was shown as the merged images (yellow). These results are the same as those untreated cells. ( F ) MCF-7 cells were treated with the IKKβ inhibitor (2 µM), fixed, and the subcellular localizations and co-localization of endogenous FOXO3 and βTrCP1 proteins were detected using Abs against FOXO3 and βTrCP1 (NIH) as described above and followed by an Alexa Fluor 594- or 488-conjugated secondary Ab, respectively, and fluorescence microscopy. DAPI was used to show the nuclei, and no co-localization (the merged yellow images) between nuclear FOXO3 and cytoplasmic βTrCP1 was detected.

Article Snippet: A specific anti-FOXO3 Ab was obtained from Imgenex Corp. Abs against β-tubulin, β-actin, hemagglutinin (HA), myc-tag and Flag-tag were purchased from Sigma.

Techniques: In Vitro, Over Expression, Incubation, Negative Control, SDS Page, Western Blot, Transfection, Control, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model

doi: 10.1371/journal.pone.0011171

Figure Lengend Snippet: ( A ) DNA samples extracted from Wt MEF or βTrCP1(–/–) MEF cells treated with camptothecin (CPT) (20 µM) for 24 or 48 h or control (DMSO) were subjected to DNA fragmentation assay. Equal amounts of the extracted DNA (2 µg/lane) and size markers (M) were subjected to electrophoresis on 2% agarose gels, which were stained with ethidium bromide and photographed. ( B ) MEF cells treated with CPT (20 µM) or DMSO (control) (0 h) for 48 h were stained with propidium iodide (PI), and the cell-cycle profiles were determined by flow cytometry. The changes in percentage of cell-cycle statuses between CPT treatment and control were shown in a histogram. ( C ) BT549 cells were transfected with βTrCP1-siRNA (si-βTrCP1) or control RNA (si-control). DNA fragmentation assay was performed as described above. ( D ) BT549 cells were transfected with control RNA (si-control) alone or transfected with FOXO3-siRNA (si-FOXO3) plus βTrCP1-siRNA (si-βTrCP1). DNA fragmentation assay was performed. ( E ) The 231 cells were transfected with βTrCP1-siRNA (designated 231-βTrCP-kd) or transfected with a βTrCP1 expression vector (designated 231-βTrCP-ov). These transfected cells or 231 control cells were injected into the nude mice as described. *, P<0.05 between 231 control versus 231-βTrCP-kd or 231-βTrCP-ov. ( F ) At 28 days after tumor cell implantation, breast tumors derived from the nude mice bearing 231 (control) or 231-βTrCP-ov or -βTrCP-kd tumors were resected, fixed, sectioned, and placed on slides. Tumor specimens were subjected to immuno-histochemical staining with an Ab specific to βTrCP1 or FOXO3. Slides were examined at 40× magnification with a microscope and representative fields are shown. Scale bars indicate 50 µm. ( G ) Total lysates prepared from 231-βTrCP-ov and 231-βTrCP-kd breast tumor specimens were subjected to IB analysis with Abs against FOXO3, βTrCP1, and β-actin (loading control). ( H ) A diagram depicts the role of βTrCP1 in promoting tumorigenesis through inducing degradation of FOXO3 protein. The subcellular localization of FOXO3 is shown to denote FOXO3 activity.

Article Snippet: A specific anti-FOXO3 Ab was obtained from Imgenex Corp. Abs against β-tubulin, β-actin, hemagglutinin (HA), myc-tag and Flag-tag were purchased from Sigma.

Techniques: Control, DNA Fragmentation Assay, Electrophoresis, Staining, Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Injection, Derivative Assay, Microscopy, Activity Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Role of STAT3-FOXO3 Signaling in the Modulation of Neuroplasticity by PD-L1-HGF-Decorated Mesenchymal Stem Cell-Derived Exosomes in a Murine Stroke Model.

doi: 10.1002/advs.202404882

Figure Lengend Snippet: Figure 4. Identification of differentially expressed genes in NPCs treated with EXO and EXO-PD-L1-HGF. a) The clustering heatmaps showed that several neurogenesis-related genes, including MET, VEGF, bFGF, CXCR4, STAT3, and FOXO3, displayed upregulation in the EXO-PD-L1-HGF treatment group. b) Comparison of the expression profiles of cells revealed that 311 genes were upregulated upon EXO-PD-L1-HGF treatment, as compared with that upon the EXO-PD-L1-HGF and control treatments, using fold-change >1.5 and an adjusted p-value < 0.05 as criteria. c) GO annotation revealed that these differentially expressed genes were involved in functions related to cell development, cell differentiation, response to stimulus, and cell motility.

Article Snippet: Antibodies against PD-L1 from OriGene Technologies; HGF, phospho-STAT3, STAT3, phospho-FOXO3, and FOXO3 from Cell Signaling Technology; and CXCR4 from Novus Biologicals; and Bcl-2, Bax, and cleaved caspase 3 from ABclonal were used to detect the respective proteins in the lysates.

Techniques: Comparison, Expressing, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Role of STAT3-FOXO3 Signaling in the Modulation of Neuroplasticity by PD-L1-HGF-Decorated Mesenchymal Stem Cell-Derived Exosomes in a Murine Stroke Model.

doi: 10.1002/advs.202404882

Figure Lengend Snippet: Figure 5. EXO-PD-L1-HGF positively regulated the STAT3/FOXO3 pathway involved in neuroprotection and neuroregeneration. a) Expression of phospho-STAT3 and phospho-FOXO3 in NPCs and PCCs upon control, EXO, and EXO-PD-L1-HGF treatments. Actin was used as the loading con- trol. b) The protein expression levels of phospho-STAT3 and phospho-FOXO3 in PCCs were decreased upon addition of a stat3 inhibitor, as observed in the western blot. c) The western blot result showed that knockdown of FOXO3 expression reduced the increased levels of phospho-FOXO3 and Bcl-2 protein expression induced by EXO-PD-L1-HGF, and enhanced the suppression of cleaved caspase-3 levels upon treatment with 300 μm H2O2. d) In vitro apoptosis results showed that knockdown of FOXO3 expression suppressed the attenuation of cell death induced by EXO-PD-L1-HGF upon treatment with 300 μm H2O2. e) The number of neurospheres formed by FOXO3-/- mice pretreated with control, EXO, and EXO-PD-L1-HGF was determined. FOXO3-/- repressed the promotion of neurosphere formation by EXO-PD-L1-HGF. f) The size of neurospheres formed by FOXO3-/- mice pretreated with control, EXO, and EXO-PD-L1-HGF was measured. FOXO3-/- mice suppressed the increase in size promoted by EXO-PD-L1-HGF. Quantified data are shown next to each graph. Data are presented as mean ± SD (*p < 0.05; **p < 0.01).

Article Snippet: Antibodies against PD-L1 from OriGene Technologies; HGF, phospho-STAT3, STAT3, phospho-FOXO3, and FOXO3 from Cell Signaling Technology; and CXCR4 from Novus Biologicals; and Bcl-2, Bax, and cleaved caspase 3 from ABclonal were used to detect the respective proteins in the lysates.

Techniques: Expressing, Control, Western Blot, Knockdown, In Vitro

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Role of STAT3-FOXO3 Signaling in the Modulation of Neuroplasticity by PD-L1-HGF-Decorated Mesenchymal Stem Cell-Derived Exosomes in a Murine Stroke Model.

doi: 10.1002/advs.202404882

Figure Lengend Snippet: Figure 7. Activation of FOXO3 modulated the EXO-PD-L1-HGF-enhanced functional recovery and infarct volume reduction poststroke. a) Immunofluo- rescence showed that EXO-PD-L1-HGF treatment promoted the differentiation of NPCs. NG2 is an oligodendrocyte marker, while GFAP is an astrocyte marker, and DCX is an immature neuron marker. b) Rotarod and c) beam walking tests result showed that knockout of FOXO3 expression (FOXO3–/–) suppressed the neurological function improvement induced by EXO-PD-L1-HGF. d) TTC staining results showed that FOXO3–/– mice repressed the attenuation of infarction size over the ischemia hemisphere. The quantified data are shown next to each graph. e) TUNEL staining was performed to detect apoptosis in the ischemia region. Cell nuclei were counterstained with DAPI (blue). Quantitative estimation of the proportion of apoptotic cells in each experimental group: LV-sh-control and LV-shFOXO3 pretreatment followed by treatment with control, EXO, and EXO-PD-L1-HGF, as indicated. Data are presented as mean ± SD (*p < 0.05; **p < 0.01). FOXO3, forkhead box 3; TTC, triphenyltetrazolium chloride; PD-L1, programmed death-ligand 1; HGF, hepatocyte growth factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4′,6-diamidino-2-phenylindole; NPCs, neural progenitor cells; GFAP, glial fibrillary acidic protein; NG2, neural/glial antigen 2; DCX, doublecortin.

Article Snippet: Antibodies against PD-L1 from OriGene Technologies; HGF, phospho-STAT3, STAT3, phospho-FOXO3, and FOXO3 from Cell Signaling Technology; and CXCR4 from Novus Biologicals; and Bcl-2, Bax, and cleaved caspase 3 from ABclonal were used to detect the respective proteins in the lysates.

Techniques: Activation Assay, Functional Assay, Marker, Knock-Out, Expressing, Staining, TUNEL Assay, Control

Journal: Biochemistry & Physiology: Open Access

Article Title: Fast Twitch Skeletal Muscle Remodeling by Prolonged Endurance Exercise is Associated with Crosstalk between Anabolic and Catabolic Signaling Pathways in Mice

doi: 10.4172/2168-9652.1000228

Figure Lengend Snippet: Figure 4: Endurance exercise potentiates anabolic signaling in TA muscle. (a) Representative images of Western blot analysis for p- AKTSer473, total AKT, p-mTORSer2448, total mTOR, p-p70S6KSer411, total p70S6K, p-ULK1Ser757, total ULK1, p-FOXO3αSer253, and total FOXO3α proteins in TA muscles of CON and EXE groups. (b-f) Quantitation of each blot. Ponceau stained proteins were used as a loading control, and thus each target protein was normalized by the loading control. Values are expressed as a mean ± SEM (n=7~9 per each group). * p<0.05 vs. CON, ** p<0.01 vs. CON. CON: sedentary control group, EXE: endurance exercise group, TA: Tibialis Anterior, SEM: Standard Error of the Mean.

Article Snippet: The antibodies used are as follows: LC3 (#4108, 1:1000), p62 (#5114, 1:1000), BNIP3 (#3769, 1:1000), AMPKα (#2532, 1:1000), p-AMPKαThr172 (#2535, 1:1000), BECLIN-1 (#3738, 1:1000), ATG7 (#2631, 1:1000), AKT (#9272, 1:1000), p-AKTSer473 (#9271, 1:1000), mTOR (#2972, 1:1000), p-mTORSer2448 (#2971, 1:1000), ULK1 (#8054, 1:1000), p-ULKSer555 (#5869, 1:1000), and p-ULK1Ser757 (#14202, 1:1000) from Cell Signaling Technology; LAMP-2 (#PA1-655, 1:1000), DRP1 (#PA1-16987, 1:500), p-DRP1Ser637 (#PA5-37534, 1:500), and OPA1 (#MA5-16149, 1:1000) from ThermoFisher Scientific; CATHEPSIN L (#133641, 1:500), MitoProfile® Total OXPHOS (#110413, 1:1000), MFN2 (#56889, 1:200), and NRF-1(#175932, 1:1000) from Abcam; BCL2 (#7382, 1:500), p-BCL2Ser87 (#377576, 1:500), p70S6K (#8418, 1:500), p-p70S6KSer411 (#8416, 1:500), and TFAM(#376672, 1:500) from Santa Cruz Biotechnology; FOXO3α (#NBP2-16521, 1:1000), pFOXO3αSer253 (#NBP1-19927, 1:1000), and PGC-1α (#NBP1-04676, 1:1000) from Novus Biologicals.

Techniques: Western Blot, Muscles, Quantitation Assay, Staining, Control