Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Activation of AQP2 by water deprivation. Changes in urinary volume ( a ) and urine osmolality ( b ) in mice at baseline and after 24 h of water deprivation. Each dot represents a mouse. * p < 0.05 (Welch’s t -test). ( c ) Western blot analysis for Ser256−phosphorylated and total AQP2 in kidney tissue from control mice and mice under water deprivation. β−actin is also shown as a loading control. ( d ) Quantification of the signal intensities expressed as a ratio of phosphorylated AQP2 to total AQP2. N = 6. *** p < 0.001 (Unpaired t -test). ( e ) Representative immunofluorescence images for Ser256−phosphorylated AQP2 (red) in kidney sections from control and water−deprived mice. The scale bar indicates 20 μm. ( f ) Quantification of the ratio of phosphorylated AQP2 signal intensities to the tubular area. The analysis was based on at least three microscopic fields of tissue from each group of mice. * p < 0.05 (Unpaired t -test).

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Uromodulin signals detected at the apical surface of the collecting duct. Representative immunofluorescence images for Ser256−phosphorylated AQP2 ( red ), uromodulin ( green ), and merged image with 4′,6−diamidino−2−phenylindole (DAPI) ( blue ) on Carnoy’s solution−fixed kidney sections from a wild−type mouse deprived of water for 24 h. Apical and cytosolic uromodulin signals were observed in the thick ascending limb of the loop of Henle (arrowheads). Uromodulin signals can be detected at the apical surface of the collecting duct (arrow). The scale bar indicates 20 μm.

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: AQP2 activation is altered in uromodulin−deficient mice. ( a ) Western blot analysis for uromodulin, Ser256−phosphorylated AQP2 and total AQP2 in kidney tissue from WT and Umod−KO mice after 24 h of water deprivation. β−actin was used as a loading control. ( b ) Quantification of signal intensities expressed as the ratio of phosphorylated AQP2 to total AQP2. n = 6. ** p < 0.01, *** p < 0.001 (Unpaired t −test). ( c ) Representative immunofluorescence images for Ser256−phosphorylated AQP2 (red) in kidney sections from WT and Umod−KO mice, with strong signals in WT mouse kidney tissue. The scale bar indicates 20 μm. ( d ) Quantification of the ratio of phosphorylated AQP2 signal intensities to the tubular area. The analysis was based on at least three microscopic fields of tissue from each group of mice. * p < 0.05 (Unpaired t −test).

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Effect of uromodulin on AQP2 activation. ( a ) Western blot analysis for Ser256-phosphorylated AQP2 and total AQP2 in lysates of mouse collecting duct cells. Cell monolayer on a filter membrane was treated with 10 −9 M dDAVP on the basolateral side and 10 −6 µg/mL uromodulin on the apical side for 24 h. β-actin was used as a loading control. ( b ) Quantification of the signal intensities expressed as a ratio of phosphorylated AQP2 to total AQP2. Analyses were based on at least three independent experiments. * p < 0.05 (Unpaired t -test).

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Activation Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Uromodulin Regulates Murine Aquaporin−2 Activity via Thick Ascending Limb–Collecting Duct Cross−Talk during Water Deprivation

doi: 10.3390/ijms23169410

Figure Lengend Snippet: Uromodulin may activate AQP2 through the suppression of endocytosis. ( a ) Representative immunofluorescence images for Ser256−phosphorylated AQP2 in mouse collecting duct cells treated with 10 −9 M dDAVP with or without 10 −6 µg/mL uromodulin and 100 µM simvastatin for 24 h. Uromodulin-treated cells showing strong signals for phosphorylated AQP2 (red). Strong linear signals for phosphorylated AQP2 at the membrane can be observed in cells treated with simvastatin, regardless of uromodulin treatment. ( b ) Intensity profiles for Ser256-phosphorylated AQP2 along axis across the cell membrane. Quantifications are based on ten axes (yellow lines) in each condition and showed as mean and SEM. Bars indicate 10 μm. DAPI, 4’,6−diamidino−2−phenylindole; umod, uromodulin; simva, simvastatin.

Article Snippet: The kidney sections were incubated with sheep anti−uromodulin (1:300; Meridian Life Science, Cincinnati, OH, USA) or rabbit anti−phospho−Ser256 AQP2 (1:300; Bioss, Woburn, MA, USA) overnight at 4 °C.

Techniques: Immunofluorescence

Journal: Cell reports

Article Title: Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria

doi: 10.1016/j.celrep.2023.113389

Figure Lengend Snippet:

Article Snippet: Phospho-FoxO1 (Ser256) Rabbit mAb , Cell Signaling Technology , Cat# 84192; RRID:AB_2800035.

Techniques: Virus, Recombinant, Staining, Bicinchoninic Acid Protein Assay, Reverse Transcription, Glucose Assay, Colorimetric Assay, Mass Spectrometry, Plasmid Preparation, Software, Transfection, Blocking Assay, Protease Inhibitor

Journal: RSC Advances

Article Title: Antidiabetic and hypolipidemic activities of eburicoic acid, a triterpenoid compound from Antrodia camphorata , by regulation of Akt phosphorylation, gluconeogenesis, and PPARα in streptozotocin-induced diabetic mice

doi: 10.1039/c8ra01841c

Figure Lengend Snippet: The expression levels of (A) p-Akt (Ser 473 )/t-Akt (Ser 473 ) and p-FoxO1 (Ser 256 )/t-FoxO1 (Ser 256 ), (B) p-AMPK (Thr 172 )/t-AMPK, PPARα, FAS, and PPARγ in the liver tissues of STZ-induced diabetic mice by oral gavage eburicoic acid (TRR). (A and B) Representative image; (C and D) quantification of the p-AMPK to t-AMPK, PPARα, FAS, and PPARγ. Protein was separated by 12% SDS-PAGE detected by western blot. All values are means ± SE ( n = 8). ### p < 0.001 compared with the control (CON) group; * p < 0.05, *** p < 0.001 compared with the streptozotocin plus vehicle (distilled water) (STZ) group. TRR1, TRR2, and TRR3, eburicoic acid (TRR) (TRR1, TRR2, and TRR3, 10, 20, and 40 mg kg −1 body weight, respectively); fenofibrate (Feno, 250 mg kg −1 body weight); metformin (Metf; 300 mg kg −1 body weight).

Article Snippet: FAS (no. 3180), phospho-Akt (Ser 473 ) (no. 4060), total AMPK (Thr 172 ), phospho-FoxO1 (Ser 256 ) (no. 11115), total-FoxO1 (Ser 256 ) (no. 2880), and β-actin (no. 4970) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, SDS Page, Western Blot, Control

Journal: BioMed research international

Article Title: Overexpression of Long Noncoding RNA H19 Downregulates miR-140-5p and Activates PI3K/AKT Signaling Pathway to Promote Invasion, Migration and Epithelial-Mesenchymal Transition of Ovarian Cancer Cells.

doi: 10.1155/2021/6619730

Figure Lengend Snippet: Figure 5: H19 overexpression activated the PI3K/AKT/mTOR pathway and the downstream protein expression in Anglne cells. (a, b) Western blot analysis of the PI3K/AKT/mTOR signaling pathway-related proteins in Anglne cells after tranfection. (c, d) Expression of the downstream proteins Forkhead box protein O1 (FoxO1), p-FoxO1 (Ser256), Murine Double Mimute 2 (MDM2), p-MDM2 (Ser166), NF-κB p65, and p-NF-κB p65 (Ser536) in Anglne cells after transfection was detected by western blot. All data were represented as mean ± S:D. ∗P < 0:05, n = 3 −4.

Article Snippet: Primary antibodies for p-mTOR (Ser2448), p-FoxO1 (Ser256), p-MDM2 (Ser166), p-NF-κB p65 (Ser536), p-GSK-3β (Ser9), PTEN, GSK-3β, mTOR, FoxO1, MDM2, and NF-κB p65 were from Santa Cruz.

Techniques: Over Expression, Expressing, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: miR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation

doi: 10.1038/cddis.2017.467

Figure Lengend Snippet: miR-181a promotes FoxO1 nuclear localization and FoxO1 acetylation in granulosa cells. ( a ) The total protein levels of FoxO1 and other proteins related to apoptosis in KGN cells were examined by western blotting after infection of Ad-miR-181a (MOI=0, 12.5, 25, and 50) for 48 h. ( b ) KGN cells were transfected with siRNA targeting FoxO1 for 12 h and then infected with Ad-miR-181a or Ad-LacZ, as indicated, for another 36 h. FoxO1, caspase-3 and cleaved caspase-3 levels were examined by western blot analysis. ( c ) KGN cells were infected with Ad-miR-181a (MOI=0, 25, and 50) for 48 h. The cytoplasmic and nuclear protein concentrations of endogenous FoxO1 were determined by western blot analysis. ( d ) An immunofluorescence assay was used to analyze the FoxO1 subcellular localization in KGN cells with the indicated treatment. FoxO1: red; DAPI: blue. ( e ) KGN cells were infected with Ad-flag-FoxO1 (MOI=20) alone or together with Ad-miR-181a as indicated for 48 h. The levels of FoxO1 acetylation and phosphorylation, total FoxO1, and SIRT1 were analyzed by western blot analysis. ( f ) KGN cells were treated with the indicated concentrations of H 2 O 2 for 12 h. The protein levels of apoptosis-related genes and FoxO1 acetylation were measured by western blot analysis. ( g ) KGN cells were infected with Ad-miR-181a (MOI=50) for 48 h or transfected with an miR-181a inhibitor (100 nM) for 36 h followed by 50 μ M H 2 O 2 treatment for another 12 h. Endogenous FoxO1 acetylation and total FoxO1 concentrations were examined by western blot analysis. ( h ) Quantification of the FoxO1 phosphorylation by making densitometric analysis on ( e )

Article Snippet: Immunoblotting was performed with primary antibodies against caspase-3 (1 : 1000; Cell Signaling Technology, #9662s, Danvers, MA, USA), cleaved caspase-3 (1 : 500; Cell Signaling Technology, #9661s), FoxO1 (1 : 1000; Cell Signaling Technology, #2880), Ac-FKHR (1 : 500; Ac-FoxO1, Santa Cruz Biotechnology, sc-49437), phospho-FoxO1 (Ser 256, 1 : 1000; Cell Signaling Technology), phospho-FoxO1 (Ser 319, 1 : 1000; Bioworld Technology, BS4712, MN, USA), FasL (1 : 500; Bioworld Technology, BS1122), Bcl-2 (1 : 1000; Proteintech, 12789-1-AP, Wuhan, China), Bax (1 : 500; Bioworld Technology, BS6420), and SIRT1 (1 : 500; Santa Cruz Biotechnology, sc-15404). β -actin (1 : 10000; Abcam, Cambridge, CA, USA), HSP60 (1 : 1000; Bioworld Technology), and Lamin B (1 : 1000; Santa Cruz Biotechnology, sc-6216) served as internal controls for detecting the expression levels of total protein lysates, cytoplasmic protein lysates, and nuclear protein lysates, respectively.

Techniques: Western Blot, Infection, Transfection, Immunofluorescence, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: miR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation

doi: 10.1038/cddis.2017.467

Figure Lengend Snippet: SIRT1 deacetylates FoxO1 in granulosa cells. ( a and b ) KGN cells were infected with or without Ad-flag-FoxO1 (MOI=20) for 12 h, followed by 10 mM NAM or 2 μ M TSA treatment for another 12 h. The levels of SIRT1, acetylated FoxO1, and total FoxO1 were measured. ( c ) KGN cells were infected with Ad-h-SIRT1 (MOI=0, 10, and 20) for 48 h. Western blotting was performed to measure the protein levels of SIRT1, acetylated FoxO1, and total FoxO1. ( d ) Western blotting was performed to analyze SIRT1, acetylated FoxO1, and total FoxO1 in KGN cells transfected with siRNA targeting SIRT1 (0, 50, or 100 nM) for 48 h. ( e ) KGN cells or mGCs were treated with H 2 O 2 at different concentrations, as indicated. The expression patterns of SIRT1, caspase-3, and cleaved caspase-3 were examined by western blot analysis. KGN cells were infected with Ad-h-SIRT1 (MOI=20) for 36 h, followed by H 2 O 2 treatment (50 μ M) for another 12 h. ( f ) Cell apoptosis was analyzed by a cell death detection assay. ** P <0.01, # P <0.05 compared with the control group. ( h ) The protein levels of SIRT1, acetylated FoxO1, total FoxO1, and apoptosis-related genes were determined by western blot analysis. KGN cells were infected with Ad-miR-181a and/or Ad-h-SIRT1, as indicated, for 48 h. ( g ) Cell apoptosis was analyzed by a cell death detection assay. ** P <0.01, # P <0.05 compared with the control group. ( i ) The protein levels of SIRT1, acetylated FoxO1, total FoxO1, and apoptosis-related genes were determined by western blot analysis

Article Snippet: Immunoblotting was performed with primary antibodies against caspase-3 (1 : 1000; Cell Signaling Technology, #9662s, Danvers, MA, USA), cleaved caspase-3 (1 : 500; Cell Signaling Technology, #9661s), FoxO1 (1 : 1000; Cell Signaling Technology, #2880), Ac-FKHR (1 : 500; Ac-FoxO1, Santa Cruz Biotechnology, sc-49437), phospho-FoxO1 (Ser 256, 1 : 1000; Cell Signaling Technology), phospho-FoxO1 (Ser 319, 1 : 1000; Bioworld Technology, BS4712, MN, USA), FasL (1 : 500; Bioworld Technology, BS1122), Bcl-2 (1 : 1000; Proteintech, 12789-1-AP, Wuhan, China), Bax (1 : 500; Bioworld Technology, BS6420), and SIRT1 (1 : 500; Santa Cruz Biotechnology, sc-15404). β -actin (1 : 10000; Abcam, Cambridge, CA, USA), HSP60 (1 : 1000; Bioworld Technology), and Lamin B (1 : 1000; Santa Cruz Biotechnology, sc-6216) served as internal controls for detecting the expression levels of total protein lysates, cytoplasmic protein lysates, and nuclear protein lysates, respectively.

Techniques: Infection, Western Blot, Transfection, Expressing, Detection Assay, Control

Journal: Cell Death & Disease

Article Title: miR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation

doi: 10.1038/cddis.2017.467

Figure Lengend Snippet: Activation of the miR-181a-SIRT1-FoxO1 apoptotic pathway in the in vivo ovarian oxidative stress model. Female 4-week-old ICR mice were injected with 50 mg/kg 3-NP ( n =8) or PBS ( n =8) twice daily for 5 days. ( a ) The miR-181a levels in ovaries were determined by qRT-PCR analysis. *** P <0.001 compared with the PBS control group. ( b ) Total protein lysates from ovarian tissue samples ( n =11) were subjected to western blot analysis and immunoprobed with antibodies specific to SIRT1, FoxO1, acetylated FoxO1, caspase-3, and cleaved caspase-3. β -actin was used as a loading control. Protein expression levels were normalized to β -actin. The data for all the samples from the PBS control group ( n =8) and the 3-NP group ( n =8) are shown in the scatter plots ( c – f ). * P <0.05, ** P <0.01 compared with the PBS control group. ( g ) Correlations between miR-181a and acetylated FoxO1 expression in GCs from the PBS and 3-NP groups. ( h ) Immunostaining for SIRT1, FoxO1, acetylated FoxO1, and cleaved caspase-3 in ovaries from the PBS and 3-NP groups. Nonspecific rabbit IgG was used as a negative control. Brown represents positive staining. Scale bar, 50 μ m

Article Snippet: Immunoblotting was performed with primary antibodies against caspase-3 (1 : 1000; Cell Signaling Technology, #9662s, Danvers, MA, USA), cleaved caspase-3 (1 : 500; Cell Signaling Technology, #9661s), FoxO1 (1 : 1000; Cell Signaling Technology, #2880), Ac-FKHR (1 : 500; Ac-FoxO1, Santa Cruz Biotechnology, sc-49437), phospho-FoxO1 (Ser 256, 1 : 1000; Cell Signaling Technology), phospho-FoxO1 (Ser 319, 1 : 1000; Bioworld Technology, BS4712, MN, USA), FasL (1 : 500; Bioworld Technology, BS1122), Bcl-2 (1 : 1000; Proteintech, 12789-1-AP, Wuhan, China), Bax (1 : 500; Bioworld Technology, BS6420), and SIRT1 (1 : 500; Santa Cruz Biotechnology, sc-15404). β -actin (1 : 10000; Abcam, Cambridge, CA, USA), HSP60 (1 : 1000; Bioworld Technology), and Lamin B (1 : 1000; Santa Cruz Biotechnology, sc-6216) served as internal controls for detecting the expression levels of total protein lysates, cytoplasmic protein lysates, and nuclear protein lysates, respectively.

Techniques: Activation Assay, In Vivo, Injection, Quantitative RT-PCR, Control, Western Blot, Expressing, Immunostaining, Negative Control, Staining

Journal: Cell Death & Disease

Article Title: miR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation

doi: 10.1038/cddis.2017.467

Figure Lengend Snippet: Knockdown of endogenous miR-181a blocked apoptotic pathway activation in the oxidative stress model. Paired ovaries of 28-day-old ICR female mice were excised, cultured in vitro , and infected with Ad-LacZ and AdmiRa-mmu-miR-181a-off adenovirus (2.5 × 10 10 pfu/ovary) for another 48 h before being stimulated with 3-NP. ( a ) qRT-PCR analysis of miR-181a expression in ovaries ( n =5 ovaries per group). *** P <0.001 compared with the Ad-LacZ+3-NP group. ( b – f ) The protein levels of SIRT1, FoxO1, acetylated FoxO1, caspase-3, and cleaved caspase-3 in ovarian tissue were determined by western blot analysis and were normalized to β -actin. * P <0.05 compared with the Ad-LacZ+3-NP group. ( g ) Immunostaining for SIRT1, FoxO1, acetylated FoxO1, and cleaved caspase-3 in cultured ovaries in vitro . Nonspecific rabbit IgG was used as a negative control. Brown represents positive staining. Scale bar, 50 μ m

Article Snippet: Immunoblotting was performed with primary antibodies against caspase-3 (1 : 1000; Cell Signaling Technology, #9662s, Danvers, MA, USA), cleaved caspase-3 (1 : 500; Cell Signaling Technology, #9661s), FoxO1 (1 : 1000; Cell Signaling Technology, #2880), Ac-FKHR (1 : 500; Ac-FoxO1, Santa Cruz Biotechnology, sc-49437), phospho-FoxO1 (Ser 256, 1 : 1000; Cell Signaling Technology), phospho-FoxO1 (Ser 319, 1 : 1000; Bioworld Technology, BS4712, MN, USA), FasL (1 : 500; Bioworld Technology, BS1122), Bcl-2 (1 : 1000; Proteintech, 12789-1-AP, Wuhan, China), Bax (1 : 500; Bioworld Technology, BS6420), and SIRT1 (1 : 500; Santa Cruz Biotechnology, sc-15404). β -actin (1 : 10000; Abcam, Cambridge, CA, USA), HSP60 (1 : 1000; Bioworld Technology), and Lamin B (1 : 1000; Santa Cruz Biotechnology, sc-6216) served as internal controls for detecting the expression levels of total protein lysates, cytoplasmic protein lysates, and nuclear protein lysates, respectively.

Techniques: Knockdown, Activation Assay, Cell Culture, In Vitro, Infection, Quantitative RT-PCR, Expressing, Western Blot, Immunostaining, Negative Control, Staining