Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

doi: 10.1186/1465-9921-14-117

Figure Lengend Snippet: Muscle protein turnover signaling is not affected following chronic LPS-treatment and GSK-3 inhibition. Protein synthesis and protein degradation-related signaling molecules were determined in whole muscle homogenates of the extensor digitorum longus (EDL) muscles by Western blot analysis of guinea pigs that were treated intranasally with LPS or SB216763 for 12 weeks. Representative immunoblots depict protein levels of (A) phospho-Akt (p-Akt), total Akt, phospho-GSK-3β (p-GSK-3β), total GSK-3β, phospho-eukaryotic initiation factor 2Bϵ (p-eIF2Bϵ), GAPDH, (B) phospho-mammalian target of rapamycin (p-mTOR), total mTOR, phospho-p70S6K (p-p70S6K), total p70S6K, phospho-S6 (p-S6), phospho-4E-BP1 (p-4E-BP1), total 4E-BP1, GAPDH, (C) phospho-FoXO1 (p-FoXO1), total FoXO1, phospho-FoXO3a (p-FoXO3a), total FoXO3a and GAPDH. The accompanying bar graphs show the densitometric analysis results (means ± SEM, n = 6), represented as a percentage of the vc control group corrected for GAPDH. All data is shown as a ratio of phospho- to total protein for each target (except p-eIF2Bϵ, p-S6 and (p-) 4E-BP1). *p < 0.05 compared with the vc control group. NS: not significant.

Article Snippet: The nitrocellulose blots were incubated overnight (4°C) with specific antibodies directed against: myosin light chains 1 (MyLC-1) and −3 (MyLC-3) (#F310) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), myosin heavy chain fast (MyHC-f) (#M4276) (Sigma-Aldrich), p-eIF2Bϵ (Ser 539 ) (#44-530G) (Invitrogen), p-mTOR (Ser 2448 ) (#2971), mTOR (#2983), p-Akt (Ser 473 ) (#9271), Akt (#9272), p-GSK-3β (Ser 9 ) (#9336), GSK-3β (#9332), p-p70S6K (Thr 389 ) (#9206), p70S6K (#2708), p-4E-BP1 (Thr 37/46 ) (#9459), 4E-BP1 (#9452), p-S6 (Ser 235/236 ) (#2211), p-FoXO1 (Ser 256 ) (#9461), FoXO1 (#2880), p-FoXO3a (Ser 253 ) (#9466), FoXO3a (#2497) and GAPDH (#2118) (all from Cell Signaling Technology), diluted in 1× TBS/0.1% Tween-20.

Techniques: Inhibition, Western Blot

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: ( A, B, C ) Representative immunoblots and densitometric analysis showed a significant increase in p-Akt Ser473 level in the ipsilateral cortex of 20 mg/kg salidroside (SALD 20)-treated injured mice at day 1 compared with vehicle-treated injured mice. The level of p-Akt Thr308 was increased following SALD 20 treatment at day 3 compared with vehicle-treated injured mice. ( D, E, F ) Representative immunoblots and densitometric analysis showed that there were no differences in the levels of p-Bad and p-FOXO1 in the ipsilateral cortex between the SALD 20 and vehicle groups at day 1 or 3. ( G, H, I ) Representative immunoblots and densitometric analysis showed that the mitochondrial Bcl-2/Bax ratio significantly increased in SALD-20 treated injured mice but no difference was found in the total Bcl-2/Bax ratio. Values are presented as mean ± SEM; # P <0.05, ## P <0.01 versus sham controls, and * P <0.05 versus vehicle-treated injured mice (n = 6 mice/group at each time point, repeated measures two-way ANOVA for p-Akt Ser473, p-Akt Thr308, p-Bad, and p-FOXO1 levels; one-way ANOVA for Bcl-2/Bax ratio).

Article Snippet: p-FOXO1 (Ser256) , Cell signaling , 9461 , Rabbit , Polyclonal , WB 1∶1000.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: Representative immunoblots and densitometric analysis showed that (A, B, C, D) levels of cleaved caspase 3, p-Akt Ser473, p-Akt Thr308, (E, F, G) p-Bad and p-FOXO1 in ipsilateral hippocampal samples from injured animals at post-injury day 1 or 3 were not significantly different from those in sham-injured animals (n = 6 mice/group at each time point, repeated measures two-way ANOVA).

Article Snippet: p-FOXO1 (Ser256) , Cell signaling , 9461 , Rabbit , Polyclonal , WB 1∶1000.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: ( A, B, C, D, E, F ) Representative immunoblots and densitometric analysis in the cortex from sham-injured mice or mice subject to cortical impact injury, infused intracerebroventricular (icv) with saline (vehicle) or LY294002. Mice received either icv pretreatment of LY294002 followed by 20 mg/kg salidroside (SALD 20) after injury (S20Y group) or icv pretreatment of saline followed by SALD 20 after CCI (S20V group). LY294002 did not affect Akt, Bad, or FOXO1 phosphorylation in sham-operated mice, but it significantly abolished SALD 20-induced preservation of p-Akt Thr308 and p-FOXO1 levels in the ipsilateral hemisphere at 1 day post-injury. The levels of p-Akt Ser473 and p-Bad were similar between the S20V and S20LY groups. Values are presented as mean ± SEM; * P <0.05, ** P <0.01 versus sham + vehicle (ShamV), and & P <0.05 versus sham + LY294002 (ShamLY), and # P <0.05 versus S20V group (n = 6 mice/group, one-way ANOVA). ( G ) Representative cresyl violet-stained brain sections of S20V and S20LY mice at 28 days post-injury. Scale bar is 1 mm. ( H ) Quantification analysis showed that there was a significant decrease in the residual cerebral tissue ratio in the S20LY group compared with the S20S group. Values are presented as mean ± SEM; * P <0.05 versus S20V group (n = 6 mice/group, Student’s t -test).

Article Snippet: p-FOXO1 (Ser256) , Cell signaling , 9461 , Rabbit , Polyclonal , WB 1∶1000.

Techniques: Western Blot, Preserving, Staining

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: Antibodies used in immunofluorescence and western blot.

Article Snippet: p-FOXO1 (Ser256) , Cell signaling , 9461 , Rabbit , Polyclonal , WB 1∶1000.

Techniques: Immunofluorescence, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

doi: 10.1155/2019/3286928

Figure Lengend Snippet: Primer sequences used for quantitative reverse transcription polymerase chain reaction.

Article Snippet: After different treatments, total protein from HK-2 cells and RPTCs was extracted and western blotting was performed as described previously [ ] using the following primary antibodies: rabbit anti-FOXO1 and TXNIP (Abcam, Cambridge, UK); rabbit anti-p-FOXO1 Ser256 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TRX, anti-FN, anti-Col IV, anti-BAX (Proteintech, Chicago, IL, USA), and anti- β -actin (Sangon Biotech, Shanghai, China).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

doi: 10.1155/2019/3286928

Figure Lengend Snippet: Tissue-specific expression of the Pax2 - Foxo1 transgene in transgenic (Tg) mice. (a) Piggy- Pax2-Foxo1 construct for the generation of Tg mice. Schematic representation of the construct containing the Pax2 promoter, CA -Foxo1 , and GFP sequences. The positions of PCR primers used to detect the transgene are shown. (b) Genomic identification of Tg mice. Foxo1 -Tg mice (T1–T9) showed a PCR-amplified 490 bp product that was undetected in non-Tg mice (N1–N2). (c) Western blot analysis of FOXO1 expression in mouse renal proximal tubule extracts of male non-Tg (N1-N3) and Tg (T1-T3) mice; β -actin was used as an internal control. (d) Immunohistochemistry staining of FOXO1 and relative staining intensity in male non-Tg and Tg mouse kidneys, using rabbit anti-FOXO1 polyclonal antibodies. Scale bars, 50 μ m.

Article Snippet: After different treatments, total protein from HK-2 cells and RPTCs was extracted and western blotting was performed as described previously [ ] using the following primary antibodies: rabbit anti-FOXO1 and TXNIP (Abcam, Cambridge, UK); rabbit anti-p-FOXO1 Ser256 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TRX, anti-FN, anti-Col IV, anti-BAX (Proteintech, Chicago, IL, USA), and anti- β -actin (Sangon Biotech, Shanghai, China).

Techniques: Expressing, Transgenic Assay, Construct, Amplification, Western Blot, Immunohistochemistry, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

doi: 10.1155/2019/3286928

Figure Lengend Snippet: Body weight, kidney weight/body weight ratio, and biochemical indicators in the normal, diabetic (DM), and Foxo1 transgenic diabetic (Tg DM) groups at week 12 after injection.

Article Snippet: After different treatments, total protein from HK-2 cells and RPTCs was extracted and western blotting was performed as described previously [ ] using the following primary antibodies: rabbit anti-FOXO1 and TXNIP (Abcam, Cambridge, UK); rabbit anti-p-FOXO1 Ser256 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TRX, anti-FN, anti-Col IV, anti-BAX (Proteintech, Chicago, IL, USA), and anti- β -actin (Sangon Biotech, Shanghai, China).

Techniques: Transgenic Assay, Injection

Journal: Oxidative Medicine and Cellular Longevity

Article Title: FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

doi: 10.1155/2019/3286928

Figure Lengend Snippet: Effect of FOXO1 on apoptosis and interstitial fibrosis in the kidneys from diabetic mice. (a) Foxo1 mRNA levels in renal proximal tubules of normal, diabetic, and diabetic Tg mice detected by qPCR at week 12; Actb was used as an internal control. (b) Representative immunoblots. (c, d) Protein levels of the total FOXO1 and p-FOXO1 detected by western blot analysis at week 12; β -actin was used as an internal control. (e–g) Foxo1 overexpression prevented the increase in the expression of fibronectin (FN), collagen IV (Col IV), and BAX proteins. All data are expressed as means ± SEM, n = 6 ( ∗ P < 0.05 vs. the normal group and # P < 0.05 vs. the diabetes group).

Article Snippet: After different treatments, total protein from HK-2 cells and RPTCs was extracted and western blotting was performed as described previously [ ] using the following primary antibodies: rabbit anti-FOXO1 and TXNIP (Abcam, Cambridge, UK); rabbit anti-p-FOXO1 Ser256 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TRX, anti-FN, anti-Col IV, anti-BAX (Proteintech, Chicago, IL, USA), and anti- β -actin (Sangon Biotech, Shanghai, China).

Techniques: Western Blot, Over Expression, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

doi: 10.1155/2019/3286928

Figure Lengend Snippet: Effect of FOXO1 on oxidative damage products and TXNIP-TRX expression in diabetic mice. (a) Effect of overexpressing Foxo1 on urinary 8-OHdG levels (ng/mg Cr) in each animal group ( n = 9). Urinary 8-OHdG levels were measured by enzyme-linked immunosorbent assay and adjusted using urinary creatine. (b) Malondialdehyde (MDA) concentrations in the kidney tissues of various groups. (c) Relative Txnip and Trx mRNA levels measured by RT-PCR. (d, e) Protein levels of TXNIP-TRX detected by western blot analysis. (f, g) Immunohistochemistry and quantitative analysis of TXNIP and TRX expression in normal, diabetic, and transgenic (Tg) diabetic mice. Data are means ± SEM, n = 6 ( ∗ P < 0.05 vs. the normal group and # P < 0.05 vs. the diabetic group).

Article Snippet: After different treatments, total protein from HK-2 cells and RPTCs was extracted and western blotting was performed as described previously [ ] using the following primary antibodies: rabbit anti-FOXO1 and TXNIP (Abcam, Cambridge, UK); rabbit anti-p-FOXO1 Ser256 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TRX, anti-FN, anti-Col IV, anti-BAX (Proteintech, Chicago, IL, USA), and anti- β -actin (Sangon Biotech, Shanghai, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Transgenic Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

doi: 10.1155/2019/3286928

Figure Lengend Snippet: TXNIP-TRX expression in HK-2 cells cultured under high glucose. (a) Expression of FOXO1 and p-FOXO1 detected by RT-PCR. FOXO1 knockin (KI) and knockout (KO) HK-2 cells were established with CRISPR/Cas9 and cultured under high glucose (HG; 4.5 g/L glucose) or normal glucose (NG; 1.0 g/L glucose) conditions. (b) Representative immunoblots. (c, d) FOXO1 and ratio of p-FOXO1/total FOXO1 as determined by densitometric analysis. (e–h) mRNA and protein levels of TXNIP and TRX detected by quantitative RT-PCR analysis of total RNA and western blot, respectively. FOXO1 KI cells were treated with the TRX inhibitor PX-12, and FOXO1 KO cells were treated with a small interfering RNA against TXNIP (si-TX). (i–k) The fibrosis- and apoptosis-related proteins, (i) FN, (j) COL IV, and (k) BAX, were detected by western blot analysis. (l–o) Chromatin immunoprecipitation assays showing FOXO1 binding to the promoter regions of TXNIP and TXN in HK-2 cells under HG. Soluble chromatin was immunoprecipitated with antibodies against FOXO1. The DNA fragments were analyzed by qPCR (l, m) or amplified by PCR and visualized on agarose gels (n, o). (p) Overexpression of FOXO1 prevents reactive oxygen species (ROS) accumulation in HG-treated HK-2 cells. Intracellular ROS production was quantified by flow cytometry analysis using 2′,6′-dichlorofluorescein diacetate. The data are presented as the means ± SEM ( n = 3). a P < 0.05 vs. normal glucose (NG); b P < 0.05 vs. HG; c P < 0.05 vs. FOXO1 knockin (KI); d P < 0.05 vs. FOXO1 knockout (KO).

Article Snippet: After different treatments, total protein from HK-2 cells and RPTCs was extracted and western blotting was performed as described previously [ ] using the following primary antibodies: rabbit anti-FOXO1 and TXNIP (Abcam, Cambridge, UK); rabbit anti-p-FOXO1 Ser256 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TRX, anti-FN, anti-Col IV, anti-BAX (Proteintech, Chicago, IL, USA), and anti- β -actin (Sangon Biotech, Shanghai, China).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Knock-In, Knock-Out, CRISPR, Western Blot, Quantitative RT-PCR, Small Interfering RNA, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Amplification, Over Expression, Flow Cytometry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: FOXO1 Overexpression Attenuates Tubulointerstitial Fibrosis and Apoptosis in Diabetic Kidneys by Ameliorating Oxidative Injury via TXNIP-TRX

doi: 10.1155/2019/3286928

Figure Lengend Snippet: FOXO1 protects against apoptosis in HK-2 cells under high glucose via regulating TXNIP-TRX. (a–h) Apoptosis was measured by flow cytometry, using FITC-annexin V and propidium iodide as markers. Data are presented as means ± SEM ( n = 3). a P < 0.05 vs. normal glucose (NG); b P < 0.05 vs. high glucose (HG); c P < 0.05 vs. FOXO1 knockin (KI); d P < 0.05 vs. FOXO1 knockout (KO).

Article Snippet: After different treatments, total protein from HK-2 cells and RPTCs was extracted and western blotting was performed as described previously [ ] using the following primary antibodies: rabbit anti-FOXO1 and TXNIP (Abcam, Cambridge, UK); rabbit anti-p-FOXO1 Ser256 (Cell Signaling Technology, Danvers, MA, USA); rabbit anti-TRX, anti-FN, anti-Col IV, anti-BAX (Proteintech, Chicago, IL, USA), and anti- β -actin (Sangon Biotech, Shanghai, China).

Techniques: Flow Cytometry, Knock-In, Knock-Out