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: BioMed Research International

Article Title: The Effects of Omega-3 Fatty Acid Supplementation on Dexamethasone-Induced Muscle Atrophy

doi: 10.1155/2014/961438

Figure Lengend Snippet: Protein expression in GA muscle samples from rats treated with n-3 PUFA or vehicle for 40 days and dexamethasone or vehicle (last 10 days). Relative optical density of total Akt (tAkt) and phosphorylated Akt (P-Akt) ((a) and (c)), total GSK3 β (tGSK3 β ) and phosphorylated GSK3 β (P-GSK3 β ) ((b) and (d)), and tAkt/P-Akt and tGSK3 β /P-GSK3 β ratios ((e) and (f), resp.). Bands representing tAkt, P-Akt, tGSK3, and P-GSK3 β are illustrated in (g). α -Tubulin (55 kDa) was used as control. The numbers represent the mean ± S.E.M. CT = control group ( n = 6), DX = dexamethasone group ( n = 5), n-3 = n-3 PUFA group ( n = 6), and DX + n-3 = dexamethasone + n-3 PUFA group ( n = 6). Values represent means ± S.E.M., analyzed by Student's t -test, # P < 0.05.

Article Snippet: The primary antibodies used were Akt (1 : 2,000, Cell Signaling #4691), P-Akt (1 : 2,000, Cell Signaling #4060, Ser473); mTOR (1 : 500, Cell Signaling #2972), P-mTOR (1 : 500, Cell Signaling #2971, Ser2448), GSK3 β (1 : 2,000, Cell Signaling #9315), P-GSK3 β (1 : 2,000, Cell Signaling #9322, Ser9), FoXO3a (1 : 500, Cell Signaling #2497), P-FoXO3a (1 : 500, Cell Signaling #9466, Ser253), and α -tubulin (1 : 30,000, Hybridoma bank) in 5% BSA/TBS-T.

Techniques: Expressing

Journal: Stem Cells International

Article Title: Intracellular Calcium Determines the Adipogenic Differentiation Potential of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells via the Wnt5a/ β -Catenin Signaling Pathway

doi: 10.1155/2018/6545071

Figure Lengend Snippet: Effect of Ca 2+ on adipogenic differentiation of MSCs-H. Cells were treated with Ca 2+ or BAPTA-AM and then cultured in adipogenic-specific medium. Adipogenic differentiation potential was monitored at day 3, day 7, day 10, and day 14, respectively. (a) Cells were stained with Oil red O, and the percentage of Oil red O-positive cells is shown (scale bar = 50 μ m, mean ± SD, n = 3, ∗∗ p < 0.01). (b) Time course of protein expression of Wnt5a, β -catenin, and p-GSK3 β in the Wnt5a/ β -catenin signaling pathway and of PPAR γ and leptin in adipogenic signaling determined by Western blot, with β -actin serving as a loading control. Expression levels were normalized to β -actin, with the expression levels in the control defined as 1 (lower panel, mean ± SD, n = 3, ∗∗ p < 0.01, ∗ p < 0.05). BAPTA: BAPTA-AM.

Article Snippet: Each membrane was incubated with antibodies against Wnt5a, β -catenin (Abcam), phospho-GSK3 β (Cell Signaling Technology, Danvers, MA, USA), PPAR γ (Santa Cruz Biotechnology), leptin (Thermo Fisher Scientific), and β -actin (Novus).

Techniques: Cell Culture, Staining, Expressing, Western Blot

Journal: Stem Cells International

Article Title: Intracellular Calcium Determines the Adipogenic Differentiation Potential of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells via the Wnt5a/ β -Catenin Signaling Pathway

doi: 10.1155/2018/6545071

Figure Lengend Snippet: Effect of Ca 2+ on the adipogenic differentiation of MSCs-L. Cells were treated with Ca 2+ and then cultured in adipogenic-specific medium. Adipogenic differentiation potential was monitored at day 3, day 7, day 10, and day 14, respectively. (a) Cells were stained with Oil red O, and the percentage of Oil red O-positive cells is shown (scale bar = 50 μ m, mean ± SD, n = 3, ∗∗ p < 0.01). (b) Time course of protein expression of Wnt5a, β -catenin, and p-GSK3 β in the Wnt5a/ β -catenin signaling pathway and of PPAR γ and leptin in the adipogenic signaling pathway determined by Western blot analysis, with β -actin serving as a loading control. Expression levels were normalized to β -actin, with the expression levels in the control defined as 1 (lower panel, mean ± SD, n = 3, ∗∗ p < 0.01, ∗ p < 0.05).

Article Snippet: Each membrane was incubated with antibodies against Wnt5a, β -catenin (Abcam), phospho-GSK3 β (Cell Signaling Technology, Danvers, MA, USA), PPAR γ (Santa Cruz Biotechnology), leptin (Thermo Fisher Scientific), and β -actin (Novus).

Techniques: Cell Culture, Staining, Expressing, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Identification and Characterization of Wnt Signaling Pathway in Keloid Pathogenesis

doi: 10.7150/ijms.5349

Figure Lengend Snippet: NF and KF cells were treated with Wnt5a (3μg/ml) for 72 hours and equal amounts of cell lysates were analyzed for total β-catenin and phosphorylated β-catenin at Ser45/Thr41 and at Ser33/37/Thr41 positions and total GSK3-β and phosphorylated GSK3-β at Ser 9 position using Western blot analyses. Both NF and KF showed a significant increase (relative to β-actin; p<0.05) in the total β-catenin (Fig. A), phosphorylated β-catenin at Ser33/37/Thr41 position (Fig. C). No significant change was found in phosphorylated β-catenin at Ser45/Thr41 (Fig. B). The Western blot bands are representative for one NF and one KF case. Data represent the mean ± SD of triplicate measurements from the three NF and three KF cases.

Article Snippet: Polyclonal rabbit antibodies were against Wnt5a (Lifespan Biosciences, Seattle, WA), phospho-beta-catenin Ser33/37/Thr41 and phospho-beta-catenin Ser45/Thr41 positions, and phospho-GSK3-beta Ser9 position (Cell Signalling Technology Inc., Beverly, MA), and monoclonal mouse antibodies were against FZD4 (R&D Systems, Minneapolis, MN), ROR2 (Santa Cruz Biotechnology, Inc., LA), beta-catenin (Upstate Biotechnology, Waltham, MA), beta-actin (Sigma-Aldrich, St. Louis, MO).

Techniques: Western Blot

Journal: International Journal of Medical Sciences

Article Title: Identification and Characterization of Wnt Signaling Pathway in Keloid Pathogenesis

doi: 10.7150/ijms.5349

Figure Lengend Snippet: NF and KF cells were treated with Wnt5a (3μg/ml) for 72 hours and equal amounts of cell lysates were analyzed for total GSK3-β and phosphorylated GSK3-β at Ser 9 position using Western blot analyses. Both NF and KF showed a significant increase (relative to β-actin; p<0.05) in the phosphorylated/inactivated GSK3-β level (Fig. B). No significant change was found in total GSK3-β (Fig. A). The Western blot band bands are representative for one NF and one KF case. Data represent the mean ± SD of triplicate measurements from the three NF and three KF cases.

Article Snippet: Polyclonal rabbit antibodies were against Wnt5a (Lifespan Biosciences, Seattle, WA), phospho-beta-catenin Ser33/37/Thr41 and phospho-beta-catenin Ser45/Thr41 positions, and phospho-GSK3-beta Ser9 position (Cell Signalling Technology Inc., Beverly, MA), and monoclonal mouse antibodies were against FZD4 (R&D Systems, Minneapolis, MN), ROR2 (Santa Cruz Biotechnology, Inc., LA), beta-catenin (Upstate Biotechnology, Waltham, MA), beta-actin (Sigma-Aldrich, St. Louis, MO).

Techniques: Western Blot

Journal: International Journal of Medical Sciences

Article Title: Identification and Characterization of Wnt Signaling Pathway in Keloid Pathogenesis

doi: 10.7150/ijms.5349

Figure Lengend Snippet: A proposed schematic representation of Wnt5a/β-catenin signaling in keloid pathogenesis. In KF, binding of the Wnt5a to FZD receptors and LRP5/6 activates Dvl which in turn inhibits β-catenin destruction complex (APC, GSK3-β, Axin) by increased phosphorylation of GSK3-β at Ser 9 position (GSK3-β inactivation) leading to the accumulation of β-catenin in the cytoplasm and translocation into the nucleus to regulate target gene transcription in cooperation with TCF and LEF family transcription factors.

Article Snippet: Polyclonal rabbit antibodies were against Wnt5a (Lifespan Biosciences, Seattle, WA), phospho-beta-catenin Ser33/37/Thr41 and phospho-beta-catenin Ser45/Thr41 positions, and phospho-GSK3-beta Ser9 position (Cell Signalling Technology Inc., Beverly, MA), and monoclonal mouse antibodies were against FZD4 (R&D Systems, Minneapolis, MN), ROR2 (Santa Cruz Biotechnology, Inc., LA), beta-catenin (Upstate Biotechnology, Waltham, MA), beta-actin (Sigma-Aldrich, St. Louis, MO).

Techniques: Binding Assay, Translocation Assay

Journal: PPAR Research

Article Title: Bezafibrate Attenuates Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis

doi: 10.1155/2017/5789714

Figure Lengend Snippet: The effect of BZA on AKT/GSK3 β , AMPK α , and MAPK signal pathways. The relative quantitative results of PPAR- α , P-AKT, P-GSK3 β , P-ERK, P-P38, and P-AMPK α in the four groups ( n = 6).

Article Snippet: The following first antibodies were purchased from Cell Signaling Technology: anti-AKT (#4691), anti-phospho-AKT (#4060), anti-GSK3 β (#9315), anti-phospho-GSK3 β (#9323P), anti-ERK (#4695), anti-phospho-ERK (#4370P), anti-P38 (#9212P), anti-phospho-P38 (#4511P), anti-AMPK α (#2603P), and anti-phospho-AMPK α (#2535).

Techniques:

Journal: PLoS ONE

Article Title: Effect of Sfrp5 on Cytokine Release and Insulin Action in Primary Human Adipocytes and Skeletal Muscle Cells

doi: 10.1371/journal.pone.0085906

Figure Lengend Snippet: Primary human adipocytes were kept untreated, exposed to Sfrp5 for 24/ml TNFα for 24 h with or without a 4 h preincubation with Sfrp5. Then, when indicated (+) cells were stimulated with insulin (10 min; 100 nM). Cell lysates were analyzed for phosphorylation of Akt-Thr308 (A), Akt-Ser473 (B), GSK3α-Ser21 (C) and PRAS40-Thr246 (D) by Western blotting. Phosphorylation signals were normalized for GAPDH protein abundance and expressed as mean ± standard error of the mean of five independent experiments using cells from different donors. The values obtained for Sfrp5-untreated, insulin-treated cells were considered as control and set at 100%. Differences among groups were calculated by two-way ANOVA followed by Bonferroni multiple comparison analysis. #, indicates p <0.05 for the effect of insulin stimulation; ***, p <0.001; **, p <0.01; *, p <0.05 versus untreated cells.

Article Snippet: Membranes were incubated with antibodies recognizing Akt-phospho-Ser473, Akt-phospho-Thr308, glycogen synthase kinase 3 (GSK3) α/β-phospho-Ser21/9, proline-rich Akt-substrate of 40-kDa (PRAS40) phospho-Thr246, NFκB-phospho-Ser536, c-jun N-terminal kinase (JNK) phospho-Thr183/Tyr185 (all from Cell Signaling Technology, Danvers, MA), Wnt5a or Sfrp5 (both from Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Effect of Sfrp5 on Cytokine Release and Insulin Action in Primary Human Adipocytes and Skeletal Muscle Cells

doi: 10.1371/journal.pone.0085906

Figure Lengend Snippet: Primary human skeletal muscle cells were kept untreated, exposed to Sfrp5 for 24/ml MCP-1 for 18 h with or without a 4 h preincubation with 100 ng/ml Sfrp5. Then, when indicated (+) cells were stimulated with insulin (10 min; 100 nM). Cell lysates were analyzed for phosphorylation of Akt-Thr308 (A), Akt-Ser473 (B), GSK3α-Ser21 (C) and PRAS40-Thr246 (D) by Western blotting. Phosphorylation signals were normalized for GAPDH protein abundance and expressed as mean ± standard error of the mean of five independent experiments using cells from different donors. The values obtained for Sfrp5-untreated, insulin-treated cells were considered as control and set at 100%. Differences among groups were calculated by two-way ANOVA followed by Bonferroni multiple comparison analysis. #, indicates p <0.05 for the effect of insulin stimulation; ***, p <0.001; **, p <0.01; *, p <0.05 versus untreated cells.

Article Snippet: Membranes were incubated with antibodies recognizing Akt-phospho-Ser473, Akt-phospho-Thr308, glycogen synthase kinase 3 (GSK3) α/β-phospho-Ser21/9, proline-rich Akt-substrate of 40-kDa (PRAS40) phospho-Thr246, NFκB-phospho-Ser536, c-jun N-terminal kinase (JNK) phospho-Thr183/Tyr185 (all from Cell Signaling Technology, Danvers, MA), Wnt5a or Sfrp5 (both from Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia

doi: 10.1155/2021/1470829

Figure Lengend Snippet: BHD activates NRF2 and promotes the phosphorylation of AKT/GSK3 β during revascularization. The protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β of tissue in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+NS group.

Article Snippet: BHD consists of seven herbal medicines which include Huang Qi, Dang Gui, Chi Shao, Chuan Xiong, Tao Ren, Hong Hua, and Di Long, with the ratio of 120 : 6 : 5 : 3 : 3 : 3 : 3, and the specific formulation was described previously [ ]; CD31 polyclonal antibody (ab28364) and VEGF polyclonal antibody (ab46154) were purchased from Abcam (UK); NAD(P)H dehydrogenase quinone 1 (NQO-1) polyclonal antibody (DF6437) was purchased from Affinity Biosciences (USA); heme oxygenase-1 (HO-1) polyclonal antibody (10701-1-AP), catalase polyclonal antibody (21260-1-AP), and histone H3 (17168-1-AP) were purchased from Proteintech (CHN); NRF2 monoclonal antibody (12721), Akt (4691) and phospho-Akt monoclonal antibody (4060), glycogen synthase kinase-3 beta (GSK3 β ) (12456) and phospho-GSK3 β (5558) monoclonal antibody, interleukin-6 (IL-6) monoclonal antibody (12912), interleukin-1beta (IL-1 β ) monoclonal antibody (12242), tumor necrosis factor- (TNF-) α monoclonal antibody (11948), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (5174) were purchased from Cell Signaling Technology (USA).

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia

doi: 10.1155/2021/1470829

Figure Lengend Snippet: BHD promotes revascularization of hindlimb of HLI mice via AKT/GSK3 β /NRF2 signal pathway. (a) The mRNA expression of NRF2 after injection of Ad-NRF2-shRNA ( n = 4). (b, c) The tissue perfusion of hindlimb in HLI mice after 0, 7, and 14 days of BHD treatment ( n = 4). (d–f) The protein expression and quantitative analysis of NRF2, VEGFA, TNF- α , IL-1 β , IL-6, CAT, NQO-1, and HO-1 and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area after 14 days of BHD treatment ( n = 4). Mean ± SD. NS, P > 0.05; ∗ P < 0.05, compared with HLI+BHD+sh-NRF2 group.

Article Snippet: BHD consists of seven herbal medicines which include Huang Qi, Dang Gui, Chi Shao, Chuan Xiong, Tao Ren, Hong Hua, and Di Long, with the ratio of 120 : 6 : 5 : 3 : 3 : 3 : 3, and the specific formulation was described previously [ ]; CD31 polyclonal antibody (ab28364) and VEGF polyclonal antibody (ab46154) were purchased from Abcam (UK); NAD(P)H dehydrogenase quinone 1 (NQO-1) polyclonal antibody (DF6437) was purchased from Affinity Biosciences (USA); heme oxygenase-1 (HO-1) polyclonal antibody (10701-1-AP), catalase polyclonal antibody (21260-1-AP), and histone H3 (17168-1-AP) were purchased from Proteintech (CHN); NRF2 monoclonal antibody (12721), Akt (4691) and phospho-Akt monoclonal antibody (4060), glycogen synthase kinase-3 beta (GSK3 β ) (12456) and phospho-GSK3 β (5558) monoclonal antibody, interleukin-6 (IL-6) monoclonal antibody (12912), interleukin-1beta (IL-1 β ) monoclonal antibody (12242), tumor necrosis factor- (TNF-) α monoclonal antibody (11948), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (5174) were purchased from Cell Signaling Technology (USA).

Techniques: Expressing, Injection, shRNA

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Buyang Huanwu Decoction Enhances Revascularization via Akt/GSK3 β /NRF2 Pathway in Diabetic Hindlimb Ischemia

doi: 10.1155/2021/1470829

Figure Lengend Snippet: BHD activates NRF2 through Akt/GSK3 β signal pathway. After injection of inhibitor, the protein expression and quantitative analysis of NRF2 in nuclear and the phosphorylation and quantitative analysis of Akt and GSK3 β in ischemic area ( n = 4). Mean ± SD. ∗ P < 0.05, compared with HLI+BHD group.

Article Snippet: BHD consists of seven herbal medicines which include Huang Qi, Dang Gui, Chi Shao, Chuan Xiong, Tao Ren, Hong Hua, and Di Long, with the ratio of 120 : 6 : 5 : 3 : 3 : 3 : 3, and the specific formulation was described previously [ ]; CD31 polyclonal antibody (ab28364) and VEGF polyclonal antibody (ab46154) were purchased from Abcam (UK); NAD(P)H dehydrogenase quinone 1 (NQO-1) polyclonal antibody (DF6437) was purchased from Affinity Biosciences (USA); heme oxygenase-1 (HO-1) polyclonal antibody (10701-1-AP), catalase polyclonal antibody (21260-1-AP), and histone H3 (17168-1-AP) were purchased from Proteintech (CHN); NRF2 monoclonal antibody (12721), Akt (4691) and phospho-Akt monoclonal antibody (4060), glycogen synthase kinase-3 beta (GSK3 β ) (12456) and phospho-GSK3 β (5558) monoclonal antibody, interleukin-6 (IL-6) monoclonal antibody (12912), interleukin-1beta (IL-1 β ) monoclonal antibody (12242), tumor necrosis factor- (TNF-) α monoclonal antibody (11948), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (5174) were purchased from Cell Signaling Technology (USA).

Techniques: Injection, Expressing

Journal: Virology Journal

Article Title: 9-aminoacridine Inhibition of HIV-1 Tat Dependent Transcription

doi: 10.1186/1743-422X-6-114

Figure Lengend Snippet: 9AA induces AKT activity and stabilization of p21WAF1 . Uninfected (CEM and Jurkat) and HIV-1 infected (ACH2 and J1.1) T-cells were treated with DMSO, 1, 2.5, or 5 μM of 9AA and collected 48 hours later. Western blot analysis was performed for Akt (pan), phospho-Akt (S473), phospho-Akt (T308), GSK3-β (pan), phospho-GSK3-β (S9), phospho-PDK1 (S241), phospho-p21WAF1 (S146), and actin.

Article Snippet: Cdk9 antibody was obtained from Biodesign International. p53, phospho-p53 (S15), AKT (pan), phospho-AKT (S473), phospho-AKT (T308), GSK3-β (pan), phospho-GSK3-β (S9) antibodies were obtained from Cell Signaling.

Techniques: Activity Assay, Infection, Western Blot