Journal: International journal of molecular medicine

Article Title: Loquat leaf extract enhances myogenic differentiation, improves muscle function and attenuates muscle loss in aged rats.

doi: 10.3892/ijmm.2015.2286

Figure Lengend Snippet: Figure 6. LE modulates Akt/mammalian target of rapamycin (mTOR) and its downstream targets in C2C12 cells. Western blot analysis of the Akt/mTOR signaling pathway in C2C12 cells. Cells were cultured with differentiation medium in the presence of LE for 24 h, harvested, lysed and processed for western blot analysis. Band density of phosphorylated proteins was quantified by densitometry and normalized to the respective unphosphorylated proteins. Fold changes are expressed relative to the untreated controls. Results are rep resentative of 3 independent experiments. LE, ethanol extract of loquat leaf; 4E‑BP1, eukaryotic translation initiation factor 4E binding protein 1.

Article Snippet: Antibodies against myosin heavy chain (MyHC; sc-20641), MyoD (sc-760), myogenin (sc-576), phosphorylated (p-)Akt (Ser473; sc-7985-R), Akt1/2/3 (sc-8312), anti-rabbit IgG-horseradish peroxidase (HRP)-conjugated antibody (sc-2054), and anti-mouse IgG-HRP-conjugated antibody (sc-2031) were obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA).

Techniques: Western Blot, Cell Culture, Binding Assay

Journal: Bone & Joint Research

Article Title: Down-expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenesis and accelerates age-related bone loss via PTEN/PI3K/AKT pathway

doi: 10.1302/2046-3758.132.bjr-2023-0146.r2

Figure Lengend Snippet: Fig. 3 miR-494-3p in MLO-Y4 cell-derived exosomes regulated osteogenic differentiation of MC3T3-E1 cells via the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/AKT pathway. a) Alkaline phosphatase (ALP) and Alizarin Red S staining of MC3T3-E1 cells on day 21 in different treatment groups. The magnification is 10× (lower part). b) and c) Western blotting and grayscale analysis of ALP, Runt-related transcription factor 2 (Runx2), collagen type I α1 (Col-I), p-AKT, AKT, PI3K, and PTEN expression in MC3T3-E1 cells in different treatment groups. Data are shown as column charts and the p-values were calculated by one-way analysis of variance with Sidak’s multiple comparison tests. The p-values were specified only when p < 0.05 (statistically significant). GD, glyceraldehyde 3-phosphate dehydrogenase; miR, microRNA; TBHP, tert-Butyl hydroperoxide.

Article Snippet: The primary antibodies used were P16 (1:1000, ab51243; Abcam, USA), Runt-related transcription factor 2 (Runx2, 1:1,000, 12,556 S; Cell Signaling Technology), collagen type I α1 (Col-I, 1:1,000, 72,026 T; Cell Signaling Technology), ALP antibody (1:1,000, AF2910; R&D Systems, USA), AKT (1:1,000, 60203-2-Ig; Proteintech, USA), phosphorylated (p)-AKT (1:1,000, 66444-1-Ig; Proteintech), PI3K (1:1,000, 20584-1-AP; Proteintech), PTEN (1:1,000, 22034-1- AP; Proteintech), CD9 (1:1,000, 60232-1-Ig; Proteintech), and CD63 (1:1,000, 67605-1-Ig; Proteintech), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5,000, 60004-1-Ig; Proteintech) was used as a loading control.

Techniques: Derivative Assay, Staining, Western Blot, Expressing, Comparison

Journal: Frontiers in Pharmacology

Article Title: Isorhamnetin Inhibits Human Gallbladder Cancer Cell Proliferation and Metastasis via PI3K/AKT Signaling Pathway Inactivation

doi: 10.3389/fphar.2021.628621

Figure Lengend Snippet: Isorhamnetin triggers cell cycle arrest in the G2/M phase and regulates PI3K/AKT cascade. (A) Cell cycle phases of NOZ and GBC-SD cells exposed to isorhamnetin (0, 40, 60, and 80 μM) for 48 h analyzed using flow cytometry. (B) Cell cycle phase distribution indicated as the mean ± SD ( n = 3). (C) Levels of expression of cyclin B1, CDK1, p27, and p53 were determined using western blot assessment. (D) The expression levels of p-PI3KP85α/γ/β-Y467/Y199/Y464, p-AKT1 (T450), PIK3CA, and AKT were detected. * p < 0.05, ** p < 0.01 relative to the control group.

Article Snippet: The membranes were then blocked using 5% skimmed milk at room temperature for 1 h. Thereafter, the membranes were incubated with the primary antibodies cleaved PARP (Cell Signaling Technology Cat# 5625, RRID:AB_10699459), BCL2 (Abcam Cat# ab32124, RRID:AB_725644), BAX (ABclonal Cat# A18642, RRID:AB_2862380), Slug (Cell Signaling Technology Cat# 9585, RRID:AB_2239535), cleaved caspases 3 (Cell Signaling Technology Cat# 9664, RRID:AB_2070042), CDK1 (Abways Cat# CY5304), cleaved caspases 9 (Cell Signaling Technology Cat# 7237, RRID:AB_10895832), cyclin B1 (ABclonal Cat# A2056), p27 (Cell Signaling Technology Cat# 3686, RRID:AB_2077850), MMP-2 (Cell Signaling Technology Cat# 13132, RRID:AB_2798128), MMP-9 (Cell Signaling Technology Cat# 13667, RRID:AB_2798289), N-cadherin (ABclonal Cat# A0433, RRID:AB_2757189), TWIST1 (ABclonal Cat# A3237, RRID:AB_2765003), P53 (ABclonal Cat# AP0263, RRID:AB_2771253), phosphorylated (p)-AKT1 (ABclonal Cat# AP0980), p-PI3KP85α/γ/β-Y467/Y199/Y464 (ABclonal Cat# AP0854, RRID:AB_2771416), E-cadherin (Cell Signaling Technology Cat# 3195, RRID:AB_2291471), PARP (Cell Signaling Technology Cat# 3195, RRID:AB_2291471), PIK3CA (ABclonal Cat# A12484, RRID:AB_2759327), AKT (Cell Signaling Technology Cat# 4691, RRID:AB_915783), caspases 9 (ABclonal Cat# A11910, RRID:AB_2758854), caspases 3 (ABclonal Cat# A2156, RRID:AB_2862975), and GAPDH (Abways Technology Cat# AB0037) at 4°C overnight.

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Isorhamnetin Inhibits Human Gallbladder Cancer Cell Proliferation and Metastasis via PI3K/AKT Signaling Pathway Inactivation

doi: 10.3389/fphar.2021.628621

Figure Lengend Snippet: Isorhamnetin inhibits GBC cell proliferation and metastasis via the PI3K/AKT pathway. After pretreatment with SC79 for 1 h, the expression levels of (A) MMP-2, Slug, MMP-9, E-cadherin, N-cadherin, TWIST1, (B) cleaved PARP, PARP, BAX, cleaved caspases 9, caspases 9, BCL-2, cleaved caspases 3, caspases 3, (C) cyclin B1, CDK1, p27, p53, p-PI3K, p-AKT1, PIK3CA, and AKT were detected and shown with statistic results. * p < 0.05, ** p < 0.01, *** p < 0.001 relative to the control group.

Article Snippet: The membranes were then blocked using 5% skimmed milk at room temperature for 1 h. Thereafter, the membranes were incubated with the primary antibodies cleaved PARP (Cell Signaling Technology Cat# 5625, RRID:AB_10699459), BCL2 (Abcam Cat# ab32124, RRID:AB_725644), BAX (ABclonal Cat# A18642, RRID:AB_2862380), Slug (Cell Signaling Technology Cat# 9585, RRID:AB_2239535), cleaved caspases 3 (Cell Signaling Technology Cat# 9664, RRID:AB_2070042), CDK1 (Abways Cat# CY5304), cleaved caspases 9 (Cell Signaling Technology Cat# 7237, RRID:AB_10895832), cyclin B1 (ABclonal Cat# A2056), p27 (Cell Signaling Technology Cat# 3686, RRID:AB_2077850), MMP-2 (Cell Signaling Technology Cat# 13132, RRID:AB_2798128), MMP-9 (Cell Signaling Technology Cat# 13667, RRID:AB_2798289), N-cadherin (ABclonal Cat# A0433, RRID:AB_2757189), TWIST1 (ABclonal Cat# A3237, RRID:AB_2765003), P53 (ABclonal Cat# AP0263, RRID:AB_2771253), phosphorylated (p)-AKT1 (ABclonal Cat# AP0980), p-PI3KP85α/γ/β-Y467/Y199/Y464 (ABclonal Cat# AP0854, RRID:AB_2771416), E-cadherin (Cell Signaling Technology Cat# 3195, RRID:AB_2291471), PARP (Cell Signaling Technology Cat# 3195, RRID:AB_2291471), PIK3CA (ABclonal Cat# A12484, RRID:AB_2759327), AKT (Cell Signaling Technology Cat# 4691, RRID:AB_915783), caspases 9 (ABclonal Cat# A11910, RRID:AB_2758854), caspases 3 (ABclonal Cat# A2156, RRID:AB_2862975), and GAPDH (Abways Technology Cat# AB0037) at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Isorhamnetin Inhibits Human Gallbladder Cancer Cell Proliferation and Metastasis via PI3K/AKT Signaling Pathway Inactivation

doi: 10.3389/fphar.2021.628621

Figure Lengend Snippet: Isorhamnetin inhibits tumor growth in vivo . (A,B) We subcutaneously administered NOZ cells into the right flank of nude mice. We then intraperitoneally administered 0.2 ml of vehicle (1% dimethyl sulfoxide and 99% corn oil) or isorhamnetin (1, 5 mg/kg) to the mice every 2 days for 14 days. Necropsy images of mice and tumors were then presented. (C,D) Tumor weight and size measured after tumor harvesting. *** p < 0.001 relative to the control group. (E) The body weight of mice was monitored during the experiment period. (F,G) Expression levels of p-PI3KP85α/γ/β, PIK3CA, cleaved PARP, PARP, cleaved caspases 9, caspases 9, p27, cleaved caspases 3, caspases 3, p53, BAX, BCL-2, N-cadherin, Slug, CDK1, p-AKT1, and AKT were detected. (H) IHC staining of Ki-67 and p-AKT1 expressions in subcutaneous xenograft tumors (magnification = 400 x) ** p < 0.01, *** p < 0.001, **** p < 0.0001 relative to the control group.

Article Snippet: The membranes were then blocked using 5% skimmed milk at room temperature for 1 h. Thereafter, the membranes were incubated with the primary antibodies cleaved PARP (Cell Signaling Technology Cat# 5625, RRID:AB_10699459), BCL2 (Abcam Cat# ab32124, RRID:AB_725644), BAX (ABclonal Cat# A18642, RRID:AB_2862380), Slug (Cell Signaling Technology Cat# 9585, RRID:AB_2239535), cleaved caspases 3 (Cell Signaling Technology Cat# 9664, RRID:AB_2070042), CDK1 (Abways Cat# CY5304), cleaved caspases 9 (Cell Signaling Technology Cat# 7237, RRID:AB_10895832), cyclin B1 (ABclonal Cat# A2056), p27 (Cell Signaling Technology Cat# 3686, RRID:AB_2077850), MMP-2 (Cell Signaling Technology Cat# 13132, RRID:AB_2798128), MMP-9 (Cell Signaling Technology Cat# 13667, RRID:AB_2798289), N-cadherin (ABclonal Cat# A0433, RRID:AB_2757189), TWIST1 (ABclonal Cat# A3237, RRID:AB_2765003), P53 (ABclonal Cat# AP0263, RRID:AB_2771253), phosphorylated (p)-AKT1 (ABclonal Cat# AP0980), p-PI3KP85α/γ/β-Y467/Y199/Y464 (ABclonal Cat# AP0854, RRID:AB_2771416), E-cadherin (Cell Signaling Technology Cat# 3195, RRID:AB_2291471), PARP (Cell Signaling Technology Cat# 3195, RRID:AB_2291471), PIK3CA (ABclonal Cat# A12484, RRID:AB_2759327), AKT (Cell Signaling Technology Cat# 4691, RRID:AB_915783), caspases 9 (ABclonal Cat# A11910, RRID:AB_2758854), caspases 3 (ABclonal Cat# A2156, RRID:AB_2862975), and GAPDH (Abways Technology Cat# AB0037) at 4°C overnight.

Techniques: In Vivo, Expressing, Immunohistochemistry

Journal: Experimental and Therapeutic Medicine

Article Title: Osthole suppresses the proliferation and induces apoptosis via inhibiting the PI3K/AKT signaling pathway of endometrial cancer JEC cells

doi: 10.3892/etm.2021.10605

Figure Lengend Snippet: Osthole exhibits anticancer effects on JEC cells through inhibiting the PI3K/AKT signaling pathway. (A) JEC cells were treated with different doses of osthole for 48 h and the expression of cleaved-caspase3, -caspase9, -PARP, Bcl-2, Bax, p-PI3K, PI3K, p-AKT and AKT were detected by Western blotting. (B) Quantitative results of indicated proteins, which were presented as the fold-change of the protein expression in the control. (C) Phospho-AKT/AKT and phospho-PI3K/PI3K were determined using ELISA. The values represent the mean ± standard error of the mean of three independent experiments. *P<0.05, **P<0.005.

Article Snippet: The primary antibodies: phosphorylated (p)-AKT (1:1,000; cat. no. 9271S); AKT (1:1,000; cat. no. 4685S); p-mTOR (1:1,000; cat. no. 2971S); and mTOR (1:1,000; cat. no. 2983S) were purchased from Cell Signaling Technology, Inc.

Techniques: Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine Reports

Article Title: miR-494-3p regulates lipopolysaccharide-induced inflammatory responses in RAW264.7 cells by targeting PTEN

doi: 10.3892/mmr.2019.10083

Figure Lengend Snippet: miR-494-3p regulates PTEN/Akt/NF-κB signaling. (A) miR-494-3p target prediction using four online tools. (B) Alignment of miR-494-3p and the 3′-UTR of PTEN. (C) Luciferase activity in 293T cells co-transfected with a reporter vector containing the 3′-UTR of PTEN and miR-494-3p mimics. RAW264.7 cells were transfected with miR-494-3p mimics, miR-494-3p inhibitor, NC or NC-i. After 24 h, cells were treated with 1 µg/ml LPS for 24 h. *P<0.05 vs. GLO-PTEN-WT. (D) PTEN expression was detected by confocal microscopy. (E) Western blot analysis was used to determine the protein expression levels of PTEN, Akt1, p-Akt1 and p65. (F) RAW264.7 cells were transfected with PTEN siRNA or control siRNA. After 48 h, cells were treated with 1 µg/ml LPS for 24 h. Western blot analysis was used to determine the protein expression levels of IL-1β, TNF-α, PTEN, Akt1, p-Akt1 and p65. (G and H) Quantification of the protein expression presented in E and F. Data are presented as the mean ± standard deviation of at least three independent experiments. *P<0.05 (miR-494-3p) vs. nc; *P<0.05 (inhibitor) vs. NC-i; *P<0.05 (si-PTEN) vs. NC. Akt1, AKT serine/threonine kinase 1; IL, interleukin; LPS, lipopolysaccharide; miR, microRNA; MT, mutant; NC, negative control; NC-i, inhibitor NC; p, phosphorylated; PTEN, phosphatase and tensin homolog; siRNA, small interfering RNA; TNF, tumor necrosis factor; UTR, untranslated region; WT, wild-type.

Article Snippet: The membrane was were blocked with 5% non-fat milk at room temperature for 1 h and incubated with primary antibodies overnight at 4°C: β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc., Danvers, MA, USA); lamina (1:1,000; 10298-1-AP; ProteinTech Group, Inc., Chicago, IL, USA); IL-1β (1:1,000; cat. no. 31202; Cell Signaling Technology, Inc.); TNF-α (1:2,000; 60291-1-Ig; ProteinTech Group, Inc.); PTEN (1:1,000; cat. no. 9552; Cell Signaling Technology, Inc.); AKT serine/threonine kinase 1 (Akt1; 1:1,000; cat. no. 2967; Cell Signaling Technology, Inc.), phosphorylated (p)-Akt1 (1:1,000; cat. no. 9018; Cell Signaling Technology, Inc.) and p65 (1:1,500; ab16502; Abcam, Cambridge, UK).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Confocal Microscopy, Western Blot, Control, Standard Deviation, Mutagenesis, Negative Control, Small Interfering RNA