Journal: Journal of Oncology

Article Title: A Water-Soluble Hydrogen Sulfide Donor Suppresses the Growth of Hepatocellular Carcinoma via Inhibiting the AKT/GSK-3 β / β -Catenin and TGF- β /Smad2/3 Signaling Pathways

doi: 10.1155/2023/8456852

Figure Lengend Snippet: Effects of HA-ADT on the apoptotic level and AKT/GSK-3 β / β -catenin pathway in human HCC cells. (a) TUNEL staining was used to detect the apoptotic level (original magnification, 100x). (b) The apoptotic index was counted as the ratio of TUNEL positive cells to total cells. (c) Flow cytometry assay was adopted to detect apoptosis. (d) Cell apoptosis distribution was analyzed. (e) Western blotting was used to determine the protein levels of AKT, p-AKT, GSK-3 β , p-GSK-3 β , β -catenin, and p- β -catenin. β -actin was adopted as the internal control. (f) The density was analyzed. All data are shown as the mean ± SEM of three independent experiments; ∗ P < 0.05, ∗∗ P < 0.01 vs. control group; △ P < 0.05, △△ P < 0.01 vs. NaHS group; ## P < 0.01 vs. GYY4137 group.

Article Snippet: The primary antibodies include anti-Cyclin E1, anti-Cyclin D1, anti-cyclin-dependent kinase (CDK) 2, anti-CDK4, anti-p27, anti-p21, anti-AKT, anti-phospho (p)-AKT (Ser473), anti-glycogen synthase kinase-3 beta (Gsk‐3 β ), anti-p-Gsk-3 β (Ser9), anti- β -catenin, anti-p- β -catenin (Ser552), anti-beclin-1, anti-p62, anti-LC3A/B, anti-Smad2, anti-p-Smad2 (Ser465/467), anti-Smad3, anti-p-Smad3 (Ser423/425), and anti-transforming growth factor-beta (TGF‐ β ) antibodies, as well as the horseradish peroxidase-conjugated secondary antibody obtained from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: TUNEL Assay, Staining, Flow Cytometry, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat

doi: 10.1016/j.jbc.2023.103059

Figure Lengend Snippet: Acute diosmin (Dios) administration improves diabetic gene programs in iWAT of mice. A , experimental model of acute control (Con) or Dios administration in mice with iWAT unilateral injection (n = 4). B , protein levels of S273 p-PPARγ, ( C ) p-IRβ, p-AKT, and p-GSK3β, ( D ) expression of gene set regulated by PPARγ S273 phosphorylation in iWAT of mice after acute Dios administration. Data are presented as mean ± SEM and ∗ p < 0.05, ∗∗ p < 0.01 compared with control group. iWAT, inguinal white adipose tissue; PPARγ, peroxisome proliferator–activated receptor γ.

Article Snippet: Membranes were incubated in 5% bovine serum albumin for 2 h and with primary antibodies overnight at 4 °C, including anti-p-PPARγ (1:2000 dilution) (catalog no.: bs-4888R; Bioss biotech), anti-PPARγ (1:1000 dilution) (catalog no.: sc-7273; Santa Cruz), anti-p-IRβ (1:2000 dilution) (catalog no.: 3025; Cell Signaling Technology), anti-p-AKT (1:2000 dilution) (catalog no.: 13038; Cell Signaling Technology), anti-p-GSK3β (1:2000 dilution) (catalog no.: 9322; Cell Signaling Technology) (1:2000 dilution), anti-UCP1 (catalog no.: Ab10983; Abcam), or β-actin (1:2000 dilution) (catalog no.: sc-47778; Santa Biotechnology).

Techniques: Injection, Expressing

Journal: Cancers

Article Title: P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

doi: 10.3390/cancers15030877

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Phospho GSK-ß 9322 , 1:1000 , Cell Signaling , LC3 ab51520 , 1:3000 , Abcam.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice

doi: 10.3389/fncel.2023.1106176

Figure Lengend Snippet: APPsα restores normal GSK3β activity and modulates the Akt/GSK3β pathway in THY-Tau22 mice. (A) Schematic overview of the regulation of GSK3β activity. Activated Akt (pAkt Ser473 ) negatively regulates the activity of GSK3β through phosphorylation of Ser 9 , which leads to GSK3β inactivation. (B) Western blot analysis of hippocampi from AAV-Venus or AAV-APPsα injected littermates (LM) or THY-Tau22 mice. Specific antibodies were used to detect total GSK3β and inactive pGSK3β Ser9 . Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (C) No differences were detected for total GSK3β between groups. (D) THY-Tau22-Venus mice revealed a strong trend toward reduced GSK3β activity, as shown by signal intensities of inactive pGSK3β Ser9 normalized to that of total GSK3β (LM-Venus vs. THY-Tau22-Venus, p = 0.060). AAV-APPsα expression restored GSK3β activity to littermate control level (THY-Tau22-Venus vs. THY-Tau22-APPsα, p = 0.051). (E) Radioactive kinase assay involving Western blot (WB) and phosphorimaging (PI) analysis after immunoprecipitation of GSK3β from AAV-Venus or AAV-APPsα injected littermates and THY-Tau22 mice. Radioactively labeled Tau was visualized using PI. Recombinant Tau (HT7), GSK3β and CDK5 were visualized by immunodetection using specific monoclonal antibodies. Note the absence of CDK5 after immunoprecipitation of GSK3β. (F) Quantitative analysis revealed significantly increased GSK3β activity (PI signal normalized to total immunoprecipitated GSK3β, WB signal) in THY-Tau22-Venus mice compared to LM-Venus mice (LM-Venus vs. THY-Tau22-Venus, ** p = 0.007). AAV-APPsα restored normal GSK3β activity (THY-Tau22-Venus vs. THY-Tau22-APPsα, ** p = 0.007). (G) Western blot analysis of total Akt and active Akt (pAkt Ser473 ) in THY-Tau22 mice after AAV-Venus or AAV-APPsα injection. Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (H,I) Quantitative analysis of the Western blot depicted in (G) . THY-Tau22 mice showed a reduction in (H) the total expression of Akt (LM-Venus vs. THY-Tau22-Venus, ** p = 0.003) and (I) for the activating Ser 473 phosphorylation of Akt (LM-Venus vs. THY-Tau22-Venus, *** p = 0.0005). AAV-APPsα rescued both total Akt and pAkt 473 (THY-Tau22-Venus vs. THY-Tau22-APPsα, *** p = 0.0002 and *** p = 0.0009), respectively. Data are depicted as mean ± SEM; N, number of animals; age of analysis, 12 months, data were analyzed using one-way ANOVA with Tukey post hoc test.

Article Snippet: The following antibodies were used: AT8 (mouse monoclonal, 1:500, #MN1020, Thermo Fisher Scientific), AT180 (mouse monoclonal, 1:1000, #MN1040, Thermo Fisher Scientific), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific), MC1 (mouse monoclonal, 1:500, kindly provided by Peter Davies), α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-p35/p25 (rabbit monoclonal, 1:1000, #2680, Cell Signaling Technology), α-Vinculin (mouse monoclonal, 1:1000, #sc-73614, Santa Cruz Biotechnology), α-GAPDH (rabbit polyclonal, 1:2000, #ABS16, Merck Millipore), α-beta-Tubulin (mouse monoclonal, 1:10000, #MAB3408, Merck Millipore), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), α-pGSK3β Ser9 (rabbit monoclonal, 1:1000, #9322, Cell Signaling Technology), α-GFAP (rabbit polyclonal, 1:1000, #173002, Synaptic Systems), α-HA-tag (mouse monoclonal, 1:1000, #2367, Cell Signaling Technology), α-PSD95 (mouse monoclonal, 1:1000, #MAB1598, Merck Millipore), α-β-catenin (mouse monoclonal, 1:500, #sc-7963, Santa Cruz Biotechnology), α-pβ-catenin Ser33/37/Thr41 (rabbit polyclonal, 1:1000, #9561, Cell Signaling Technology), M3.2 (mouse monoclonal, 1:1000, a kind gift from Paul Mathews), α-Akt (rabbit monoclonal, 1:1500, #4691, Cell Signaling Technology), α-pAkt Ser473 (rabbit monoclonal, 1:1000, #4060, Cell Signaling Technology).

Techniques: Activity Assay, Western Blot, Injection, Staining, Expressing, Kinase Assay, Immunoprecipitation, Labeling, Recombinant, Immunodetection

Journal: Disease Markers

Article Title: Leptin Promotes HTR-8/SVneo Cell Invasion via the Crosstalk between MTA1/WNT and PI3K/AKT Pathways

doi: 10.1155/2022/7052176

Figure Lengend Snippet: Leptin mediates β -catenin activation through the crosstalk between MTA1/WNT and PI3K/AKT pathways in HTR-8/SVneo cells. (a) HTR-8/SVneo cells were treated with exogenous leptin (0 and 200 ng/ml) for 24 h, and MTA1, WNT1, p-GSK3 β (Ser9), and p-AKT (Ser473) levels were detected by Western blot. Data are shown as mean ± SD; ∗ P < 0.01 vs. leptin (0 ng/ml). (b) The knockdown efficiencies of MTA1 and WNT1 were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (c) HTR-8/SVneo cells were transfected with MTA1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of MTA1, WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (d) HTR-8/SVneo cells were transfected with WNT1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (e) The knockdown efficiencies of AKT and β -catenin were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (f) HTR-8/SVneo cells were transfected with AKT siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of AKT, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. All experiments were performed in triplicate.

Article Snippet: The following primary antibodies were used: MMP9 (ab76003), MTA1 (ab71153), WNT1 (ab15251), AKT (ab179463), GSK3 β (ab32391), β -catenin (ab32572), histone 3 (ab1791), β -actin (ab6276) (Abcam, USA); p-AKT (Ser473) (#4060), p-GSK3 β (Ser9) (#9322) (Cell Signaling Technology, USA).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Transfection, Expressing

Journal: Cell Death Discovery

Article Title: Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals

doi: 10.1038/s41420-022-01291-z

Figure Lengend Snippet: A , B The activation level of AKT/GSK3β pathway was detected by western blot in vitro and in vivo. C , D The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1), inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) and fibrosis marker molecules (COL-1, COL-3 and α-SMA) was detected by western blot. E Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ** P < 0.01 and *** P < 0.001. F The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.

Article Snippet: The information of antibodies is as follows: IGF1 (1: 1000, Abclonal, #A11985, China), IGF1R (1: 1000, CST, #9750, USA), p-IGF1R(Tyr1135/1136) (1: 1000, CST, #3024, USA), COL-1 (1: 2000, Abcam, ab260043, UK), COL-3 (1: 1000, Abcam, ab184993, UK), α-SMA (1:1000, CST, #19425, USA), heme oxygenase-1 (HO-1, 1: 1000, CST, #5853, USA), N-cadherin (1: 1000, CST, #49398, USA), E-cadherin (1: 1000, CST, #49398, USA), TGFβ1 (1: 1000, abclonal, #A2124, China), p-NF-kB p65(Ser536) (1: 1000, CST, #3033, USA), NF-kB p65 (1: 1000, CST, #8242, USA), NLRP3 (1: 1000, CST, #15101, USA), Cleaved Caspase1(Asp297) (1: 1000, CST, #4199, USA), Caspase1 (1: 1000, CST, #2225, USA), p-Akt(Ser473) (1: 1000, CST, #4060, USA), Akt (1: 1000, CST, #4691, USA), p-GSK3β(Ser9) (1: 1000, CST, #9322, USA), GSK3β (1: 1000, CST, #9369, USA) and β-actin (1: 1000, CST, #4970, USA).

Techniques: Activation Assay, Western Blot, In Vitro, In Vivo, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Effect of repeated intranasal LPS challenge and treatment with the selective GSK-3 inhibitor SB216763 on extracellular matrix deposition in the lung. (A) Expression of fibronectin was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. Effects of repeated LPS challenge and SB216763 treatment on fibronectin expression were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (B) Histological staining of the extracellular matrix protein collagen using Sirius Red. The non-cartilaginous airways were digitally photographed (100-200 × magnification) and analysed by using ImageJ software. Effects of repeated LPS challenge and SB216763 treatment on airway collagen expression were quantified, representing mean ± s.e.m. of 9 animals per group. (C) The mean linear intercept (LMI), a measure for alveolar airspace size, was determined by staining the tissue-sections with haematoxylin and eosin. Data represent means ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Staining, Software

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Repeated LPS instillation and pharmacological inhibition of GSK-3 by SB216763 do not affect airway smooth muscle content. Immunohistological analysis of sm-MHC positive area in (A) large (cartilaginous) and (B) small (non-cartilaginous) airways. Effects of repeated LPS challenge and SB216763 treatment on airway smooth muscle sm-MHC expression were quantified. Data represent means ± s.e.m. of 9 animals per group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Right ventricle hypertrophy after repeated intranasal LPS instillation is attenuated by GSK-3 inhibition. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on right ventricle hypertrophy. Effects of repeated LPS challenge and SB216763 treatment on size of right ventricle were quantified as right ventricle weight over total heart weight, representing mean ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: GSK-3 inhibition does not inhibit LPS-induced pulmonary inflammation. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on inflammatory cell infiltration in the airways. Cells within 50 μm of the basement membrane were quantified and expressed relative to basement membrane length, representing mean ± s.e.m. of 9 animals per group. *p < 0.05 compared to control group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Activation of β-catenin in response to repeated intranasal LPS challenge is prevented by treatment with the selective GSK-3 inhibitor SB216763. (A) Expression of active β-catenin, phosphorylated GSK-3 (ser9/21 GSK-3) and total GSK-3 was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. (B,C) Responses of repeated LPS challenge and SB216763 treatment on active β-catenin expression (B) and GSK-3 phosphorylation (C) were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (D) Correlation between pulmonary expression of fibronectin (data from Figure ) and active β-catenin in all guinea pigs. R = 0.552; p < 0.001. (E) Immunofluorescence analysis of active β-catenin (red) in large airways counterstained with Hoechst 3342 to stain nuclei (blue). *p < 0.05 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining