Journal: Molecular Medicine Reports

Article Title: Activation of AKT1 enhances the capacity of senescent BMSCs to regulate osteoclast activation

doi: 10.3892/mmr.2025.13642

Figure Lengend Snippet: Changes in the AKT1 expression level in replicative-aged BMSCs. (A) SA-β-gal staining to observe cellular senescence. Scale bar, 75 µm. (B) WB detection of the protein expression levels of the cellular senescence markers, p21 and p16, in the P10-MSCs group and P3-MSCs group. (C) WB and PCR were used to detect the relative AKT1 protein and mRNA levels, respectively. **P<0.01, ***P<0.001, ****P<0.0001. BMSCs, bone mesenchymal stromal cells; SA-β-gal, senescence-associated β-galactosidase; WB, western blotting.

Article Snippet: After adhesion, transfection complexes were prepared by adding 15 μg AKT1 cDNA recombinant plasmid [pcDNA3.1(+); plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) containing mouse AKT1 ORF sequence (cat. no. MG50254-M; Sino Biological Inc.)] to 300 μl Opti-MEM I in one tube and 30 μl Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to 300 μl Opti-MEM I in another, incubating both for 5 min, combining them, incubating for 10 min and adjusting the volume to 6 ml with Opti-MEM I.

Techniques: Expressing, Staining, Western Blot

Journal: Molecular Medicine Reports

Article Title: Activation of AKT1 enhances the capacity of senescent BMSCs to regulate osteoclast activation

doi: 10.3892/mmr.2025.13642

Figure Lengend Snippet: Overexpression efficiency of AKT1 was detected by WB and PCR. (A) Detection of the protein expression levels of AKT1, p16 and p21 through WB. (B) AKT1 protein expression in the four groups (P10-MSCs, P3-MSCs, P10-MSCs + pcDNA NC and P10-MSCs + pcDNA AKT1). (C) AKT1 mRNA expression in the four groups (P10-MSCs, P3-MSCs, P10-MSCs + pcDNA NC and P10-MSCs + pcDNA AKT1). *P<0.05, ****P<0.0001. WB, western blotting.

Article Snippet: After adhesion, transfection complexes were prepared by adding 15 μg AKT1 cDNA recombinant plasmid [pcDNA3.1(+); plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) containing mouse AKT1 ORF sequence (cat. no. MG50254-M; Sino Biological Inc.)] to 300 μl Opti-MEM I in one tube and 30 μl Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to 300 μl Opti-MEM I in another, incubating both for 5 min, combining them, incubating for 10 min and adjusting the volume to 6 ml with Opti-MEM I.

Techniques: Over Expression, Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: Activation of AKT1 enhances the capacity of senescent BMSCs to regulate osteoclast activation

doi: 10.3892/mmr.2025.13642

Figure Lengend Snippet: Effect of AKT1 overexpression on the replicative senescence of BMSCs. (A) SA-β-gal staining to observe cellular senescence. Scale bar, 75 µm. (B and C) Protein expression of the cell ageing markers, p21 and p16, in the four groups (P10-MSCs, P3-MSCs, P10-MSCs + pcDNA NC and P10-MSCs + pcDNA AKT1). **P<0.01, ***P<0.001, ****P<0.0001. BMSCs, bone mesenchymal stromal cells; SA-β-gal, senescence-associated β-galactosidase.

Article Snippet: After adhesion, transfection complexes were prepared by adding 15 μg AKT1 cDNA recombinant plasmid [pcDNA3.1(+); plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) containing mouse AKT1 ORF sequence (cat. no. MG50254-M; Sino Biological Inc.)] to 300 μl Opti-MEM I in one tube and 30 μl Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to 300 μl Opti-MEM I in another, incubating both for 5 min, combining them, incubating for 10 min and adjusting the volume to 6 ml with Opti-MEM I.

Techniques: Over Expression, Staining, Expressing

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: AKT1 increases the phosphorylation of Kaiso at T606. (A ) A conservative RSX T XP motif within Kaiso of human and other species ( B ) After starvation overnight, treatments of Insulin (100 ng/mL), IL-6 (10 ng/mL) and fetal bovine serum (FBS, 1:10000 v/v) for 15 min increased the phosphorylation level of endogenous Kaiso in MGC803 cells. ( C ) Effects of AKT inhibitor MK2206 treatment (10 μmol/mL) for 30 min blocked the promotion of Insulin induced Kaiso phosphorylation as pAKT substrate in MGC803 cells. ( D ) AKT1 overexpression at different doses increased the amount of phosphorylated AKT substrate (pAKT-sub) in Kaiso complexes immunoprecipitated by Kaiso antibody in MGC803 cells. ( E ) The activity comparison of three kinase candidates to phosphorylate Kaiso at T606 in MGC803 and BGC823 cells. ( F ) The T606-phosphorylation status of endogenous Kaiso in MGC803 and BGC823 with AKT1 overexpression after starvation overnight.

Article Snippet: Plasmid pEBG-GST-Kaiso was generated by inserting the full-length Kaiso coding sequence into BamH I and Not I sites of pEBG vector. pcDNA3.1-HA-AKT1 vector was purchased from Addgene (#9008, MA, USA); pEZ-M56-14-3-3γ-mCherry, pEZ-M56-14-3-3σ-mCherry vectors, from FulenGen (EX-T4084-M56, EX-T4084-M98-5, EX-C0507-M98, Guangzhou, China); pENTER-Flag-14-3-3 isoforms (α/β, ε, η, δ/γ, τ, ζ, σ) were purchased from Vigene Bioscience (CH867785, CH897212, CH845486, CH898602, CH878525, CH824520, CH890307, Shandong, China); Insulin (P3376, Beyotime, Shanghai, China), IL-6 (Cat. 200-06, Proteintech, NJ, USA), EGF (PHG6045, Thermo Fisher Scientific, MA, USA), MK2206 (HY-10358, MedChemExpress, NJ, USA) were also used in the study.

Techniques: Over Expression, Immunoprecipitation, Activity Assay

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: AKT1 and 14-3-3 regulate the T606-phosphorylation and subcellular localization of endogenous Kaiso. (A ) Endogenous Kaiso in MGC803 cells immunoprecipitated by Kaiso antibody was identified by the antibody specific for AKT substrate motif, and the immunoprecipitation by AKT substrate antibody was identified by antibody against Kaiso. ( B ) AKT1 overexpression increased endogenous Kaiso-14-3-3 interaction in MGC803 cells in Co-IP assay. ( C and D ) The T606-phosphorylation states of endogenous Kaiso in the cytoplasm and nucleus of MGC803 and BGC823 cells with 14-3-3σ and 14-3-3γ overexpression, respectively.

Article Snippet: Plasmid pEBG-GST-Kaiso was generated by inserting the full-length Kaiso coding sequence into BamH I and Not I sites of pEBG vector. pcDNA3.1-HA-AKT1 vector was purchased from Addgene (#9008, MA, USA); pEZ-M56-14-3-3γ-mCherry, pEZ-M56-14-3-3σ-mCherry vectors, from FulenGen (EX-T4084-M56, EX-T4084-M98-5, EX-C0507-M98, Guangzhou, China); pENTER-Flag-14-3-3 isoforms (α/β, ε, η, δ/γ, τ, ζ, σ) were purchased from Vigene Bioscience (CH867785, CH897212, CH845486, CH898602, CH878525, CH824520, CH890307, Shandong, China); Insulin (P3376, Beyotime, Shanghai, China), IL-6 (Cat. 200-06, Proteintech, NJ, USA), EGF (PHG6045, Thermo Fisher Scientific, MA, USA), MK2206 (HY-10358, MedChemExpress, NJ, USA) were also used in the study.

Techniques: Immunoprecipitation, Over Expression, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation

doi: 10.1038/s41598-017-05564-x

Figure Lengend Snippet: Interaction of ASXL1 with AKT1 and its role in AKT1 activation. ( a ) Schematic representation of mouse Asxl1 and human AKT1. Three conserved ASXN, ASXM, and PHD domains from the ASXL family and NR box (LRMLL) are indicated by closed boxes ( left ). The three conserved PH, kinase, and regulatory domains (closed boxes) among the AKT family and the phosphorylated site are shown ( right ). ( b ) Endogenous interaction between ASXL1 and AKT1. Cell lysates from H1299 cells were subjected to IP using anti-AKT1 or anti-ASXL1 antibody, and the precipitated proteins were visualized by WB with anti-ASXL1 or anti-AKT1 antibody. Star mark indicates non-specific band . ( c ) Effects of kinase activity on the interaction. H1299 cells were transfected with Flag-AKT1, constitutively active (CA), or kinase-deficient (KD) mutant expression vectors, and cell lysates were prepared for IP and WB analysis. ( d ) Mapping of ASXL1 domain responsible for AKT1 binding. GST pull-down assays were performed using purified His-AKT1 and GST-ASXL1 fragments. ( e ) Cytoplasmic interaction between ASXL1 and AKT1. Either nuclear or cytoplasmic fractionation of H1299 cells was determined by WB using indicated antibodies (T, total; C, cytoplasmic; N, nuclear fraction). LSD1 and β-actin serve as controls for nuclear and cytosolic fractions, respectively. ( f ) Effect of Asxl1 disruption on AKT1 phosphorylation. WB analysis was performed using MEFs from Asxl1 +/+ or Asxl1 −/− mice in the absence or presence of IGF-1 for 30 min and anti-p-Akt1 antibody.

Article Snippet: The desired AKT1 variants were created by PCR amplification using AKT1 cDNA (Sino Biological, North Wales, PA) and subcloned into suitable vectors (Flag (2×)-tagged and Myc-tagged pcDNA3 vectors) for overexpression in mammalian cells.

Techniques: Activation Assay, Activity Assay, Transfection, Mutagenesis, Expressing, Binding Assay, Purification, Fractionation

Journal: Scientific Reports

Article Title: Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation

doi: 10.1038/s41598-017-05564-x

Figure Lengend Snippet: Effect of Asxl1 disruption on p27Kip1 phosphorylation and Rb activation. ( a ) Hypophosphorylation of p27Kip1 by Asxl1 disruption. Phosphorylation of p27Kip1 (Thr157) was monitored by WB using an anti-p-p27Kip1 antibody. ( b ) Ternary complex of ASXL1, AKT1, and p27Kip1. Lysates were prepared from HEK293 cells with or without IGF-1 treatment and subjected to IP using an anti-p27 antibody. The precipitated proteins were analyzed by WB using anti-ASXL1 and anti-AKT antibody. ( c ) Effect of Asxl1 disruption on the subcellular localization of p27Kip1. Fluorescence microscopy was performed with MEFs using primary anti-p27 antibody and Alexa 488-conjugated secondary antibody. Hoechst staining was used to visualize chromosomal DNA. ( d ) Hypo-phosphorylation of Rb. The Rb phosphorylation (Ser807/811) of MEFs (passage 5) from Asxl1 +/+ or Asxl1 −/− mice was monitored daily by WB using an anti-p-Rb antibody. ( e , f ) Increased Rb binding to the Ccna2 promoter upon Asxl1 deletion. ChIP assays with anti-E2F1 ( e ) or anti-Rb ( f ) antibody and primer set for mouse Ccna2 promoter (Supplementary Table ). Asxl1 +/+ and Asxl1 −/− mice-derived MEFs were treated with IGF-1. Chromatin binding was represented as a percentage of input. Data are the mean ± SD of three independent experiments (* p < 0.05).

Article Snippet: The desired AKT1 variants were created by PCR amplification using AKT1 cDNA (Sino Biological, North Wales, PA) and subcloned into suitable vectors (Flag (2×)-tagged and Myc-tagged pcDNA3 vectors) for overexpression in mammalian cells.

Techniques: Activation Assay, Fluorescence, Microscopy, Staining, Binding Assay, Derivative Assay

Journal: Scientific Reports

Article Title: Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation

doi: 10.1038/s41598-017-05564-x

Figure Lengend Snippet: Functional correlation between Asxl1 disruption and AKT1 inhibition. ( a , b ) The induction of senescence by an AKT1 inhibitor. Wild-type (WT) and Asxl1 -null MEFs were treated with IGF-1 alone or IGF-1 plus AKT inhibitor IV (iAKT) and subjected to senescence assays by SA-β-gal staining and SAHF formation. The numbers of β-gal stained cells ( a ) or cells with heterochromatin foci ( b ) were counted. Data are the mean ± SD of three independent experiments (* p < 0.05). ( c ) The effect of the AKT1 inhibitor on the subcellular localization of p27Kip1. MEFs were treated with IGF-1 alone or IGF-1 plus iAKT and subjected to immunofluorescence microscopy using anti-p27 antibody and Alexa 488-conjugated secondary antibody.

Article Snippet: The desired AKT1 variants were created by PCR amplification using AKT1 cDNA (Sino Biological, North Wales, PA) and subcloned into suitable vectors (Flag (2×)-tagged and Myc-tagged pcDNA3 vectors) for overexpression in mammalian cells.

Techniques: Functional Assay, Inhibition, Staining, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: Asxl1 deficiency in embryonic fibroblasts leads to cellular senescence via impairment of the AKT-E2F pathway and Ezh2 inactivation

doi: 10.1038/s41598-017-05564-x

Figure Lengend Snippet: Hypothetical model for the role of ASXL1 in cell cycle progression through the cooperation with AKT1 and EZH2. For details, see the Discussion section.

Article Snippet: The desired AKT1 variants were created by PCR amplification using AKT1 cDNA (Sino Biological, North Wales, PA) and subcloned into suitable vectors (Flag (2×)-tagged and Myc-tagged pcDNA3 vectors) for overexpression in mammalian cells.

Techniques: