Journal: Acta Pharmacologica Sinica

Article Title: Tubeimoside-1, a triterpenoid saponin, induces cytoprotective autophagy in human breast cancer cells in vitro via Akt-mediated pathway

doi: 10.1038/s41401-018-0165-9

Figure Lengend Snippet: TBMS1 inhibits Akt activity. a The prediction statistics (accuracy and AUC) of eight SAR models for eight autophagy-related proteins. b SPRi graph showing the interaction of TBMS1 and Akt1. c MDA-MB-231, MCF-7, or T47D cells were treated with a series of concentrations of TBMS1 for 24 h, and the levels of p-Akt (S473, T308) and Akt were measured by Western blot. β-Actin was used as a loading control

Article Snippet: Recombinant human Akt1 protein (cat. no. 10763-H08B) was purchased from Sino Biological (Beijing, China).

Techniques: Activity Assay, Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: Tubeimoside-1, a triterpenoid saponin, induces cytoprotective autophagy in human breast cancer cells in vitro via Akt-mediated pathway

doi: 10.1038/s41401-018-0165-9

Figure Lengend Snippet: The kinetic parameters of TBMS1 and Akt1 binding from SPRi

Article Snippet: Recombinant human Akt1 protein (cat. no. 10763-H08B) was purchased from Sino Biological (Beijing, China).

Techniques: Binding Assay

Journal: Scientific reports

Article Title: The osteogenesis-promoting effects of alpha-lipoic acid against glucocorticoid-induced osteoporosis through the NOX4, NF-kappaB, JNK and PI3K/AKT pathways.

doi: 10.1038/s41598-017-03187-w

Figure Lengend Snippet: Figure 5. The protein expression levels of NOX4, NF-kappaB, JNK and the PI3K/AKT signalling pathway on femur. Nox4 (A), levels of phosphorylation of p65 (B), levels of phosphorylation of JNK (C) and levels of phosphorylation of AKT (D) were detected by western blotting. Data were the means ± SD (n = 6 for each group). #p < 0.05 and ##p < 0.01 compared with the SHAM group. *p < 0.05 and **p < 0.01 compared with the DEX group. GAPDH was used as loading control.

Article Snippet: Antibodies specific for Bcl-2 (Catalog No. 12789-1-AP), caspase-9 (Catalog No. 10380-1-AP), caspase-3 (Catalog No. 19677-1-AP), NOX4 (Catalog No. 14347-1-AP), JNK (Catalog No. 51151-1-AP), and AKT (Catalog No. 10176-2-AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: Scientific reports

Article Title: The osteogenesis-promoting effects of alpha-lipoic acid against glucocorticoid-induced osteoporosis through the NOX4, NF-kappaB, JNK and PI3K/AKT pathways.

doi: 10.1038/s41598-017-03187-w

Figure Lengend Snippet: Figure 9. The expression levels of NOX4, NF-kappaB, JNK and the PI3K/AKT signalling pathway in MC3T3-E1 cells. NOX4 (A) and levels of phosphorylation of p65 (B) and levels of phosphorylation of JNK (C) and levels of phosphorylation of AKT (D) were detected by western blotting. GAPDH was used as loading control. Data were expressed as mean ± SD of three independent experiments. #p < 0.05 and ##p < 0.01 compared with the control group. *p < 0.05 and **p < 0.01 compared with the DEX-alone group.

Article Snippet: Antibodies specific for Bcl-2 (Catalog No. 12789-1-AP), caspase-9 (Catalog No. 10380-1-AP), caspase-3 (Catalog No. 19677-1-AP), NOX4 (Catalog No. 14347-1-AP), JNK (Catalog No. 51151-1-AP), and AKT (Catalog No. 10176-2-AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Overexpression of WDFY2 inhibits prostate cancer cell growth and migration via inactivation of Akt pathway.

doi: 10.1177/1010428317704821

Figure Lengend Snippet: Figure 4. WDFY2 influenced prostate cancer development via Akt pathway specifically. (a) and (b) Western blotting showed that overexpressing WDFY2 did not affect the expression of E-cadherin and SLUG. (c) Real-time RT-PCR analysis of the mRNA level of Snail, Slug, Twist, ZEB1, and ZEB2 in the DU145 cells transfected with control vector and WDFY2. (d) and (e) WDFY2 overexpression showed specific downregulation of pAkt (Ser473). The values were the mean ± SEM of three independent experiments, and the p value is shown from Student’s t-test analysis.

Article Snippet: Some manufacturers were responsible for the production of antibodies, including anti-WDFY2 antibody (SAB), anti-E-cadherin (610181; BD Transduction Laboratories), anti-phospho-GSK3β, anti-Akt, anti-phospho-Akt (Ser473), anti-STAT3, anti-pSTAT3 (Tyr705), anti-P65, anti-pP65 (Ser536), anti-b-actin (Sigma-Aldrich), and also anti-tubulin (Proteintech).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Over Expression

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A,B) Western blots showing the effect of ATP for 30 minutes on AKT and PTEN phosphorylation in normal ciliated cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88), and the iCCA cell line HUCTT1. Bar graph shows densitometry expressed as % of phosphorylated/total ratios in control conditions (*p<0.05, n=3) (**p<0.01, n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: Nucleotides are detected by P2Y11 receptor localized in the primary cilium activating AC5, which induces increases in the levels of cAMP and activation of PKA. Once activated PKA phosphorylates and activates LKB1, LKB1 phosphorylates and stabilizes PTEN, leading to AKT inhibition. Inhibited AKT is unable to phosphorylate F-actin that is required to reorganize the cytoplasm and protrusions in migrating cells. HMC can activate LKB1 via a ciliary-independent mechanism emulating the chemosensory function of primary cilia.

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Activation Assay, Inhibition

Journal: Journal of Cellular and Molecular Medicine

Article Title: The preventive and therapeutic effects of AAV1‐KLF4‐shRNA in cigarette smoke‐induced pulmonary hypertension

doi: 10.1111/jcmm.16194

Figure Lengend Snippet: (A) Expression of KLF4 in the pulmonary vessels of rats in each group. Scale bar: 30 μm (B) Expression of PCNA in the pulmonary vascular smooth muscle of rats in each group. Scale bar: 25 μm (C) Representative bands of protein expression for KLF4 and PCNA in rat pulmonary vessels, and comparison of relative protein levels (normalized to GAPDH level). AU, arbitrary units. (D) Expression of P‐AKT in the pulmonary vessels of rats in each group. Scale bar: 20 μm. Sham (n = 8), saline (n = 6), AAV1‐control vector group (n = 6) and AAV1‐KLF4‐shRNA group (n = 11) at the end of the prevention experiment. Multiple comparisons were performed by one‐way ANOVA with SNK‐q. ** P < .01, *** P < .001

Article Snippet: Furthermore, the expression of KLF4 and P‐AKT was assessed by immunostaining with antibodies against KLF4 (ProteinTech, Wuhan, China) and P‐AKT (Gene Tex, San Antonio, USA).

Techniques: Expressing, Plasmid Preparation, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: The preventive and therapeutic effects of AAV1‐KLF4‐shRNA in cigarette smoke‐induced pulmonary hypertension

doi: 10.1111/jcmm.16194

Figure Lengend Snippet: (A) Expression of KLF4 in the pulmonary vessels of rats in each group. Scale bar: 30 μm (B) Expression of PCNA in the pulmonary vascular smooth muscle of rats in each group. Scale bar: 25 μm (C) Representative bands of protein expression for KLF4 and PCNA in rat pulmonary vessels, and comparison of relative protein levels (normalized to GAPDH level). AU, arbitrary units. (D) Expression of P‐AKT in the pulmonary vessels of rats in each group. Scale bar: 20 μm. Sham (n = 6), saline (n = 7), AAV1‐control vector group (n = 7) and AAV1‐KLF4‐shRNA group (n = 8) at the end of the therapeutic experiment. Multiple comparisons were performed by one‐way ANOVA with SNK‐q. * P < .05, ** P < .01, *** P < .001

Article Snippet: Furthermore, the expression of KLF4 and P‐AKT was assessed by immunostaining with antibodies against KLF4 (ProteinTech, Wuhan, China) and P‐AKT (Gene Tex, San Antonio, USA).

Techniques: Expressing, Plasmid Preparation, shRNA

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) Transgenic mice expressing a doxycycline-inducible cyclin D1 WT and cyclin D1 KE mutant cDNA targeted to the mammary gland by MMTV-RtTA were treated with doxycycline for 14 days and immunohistochemical analysis was conducted for phosphorylated targets of Akt1. (B–D) Representative immunohistochemistry (IHC) results are shown, and quantitative data are indicated as mean ± SEM at the right of the panels. IHC was conducted for (B) cyclin D1 cDNA (FLAG), (C) phospho-Akt1 Ser473, and (D) Akt1. (E) The cyclin D1 fl/fl mice were intercrossed with ROSA26-ER-Cre, and the mice treated with tamoxifen for 5 days. Immunohistochemical staining of the mammary epithelium was conducted. (F–H) Immunohistochemical staining of the mammary gland for cyclin D1 and Akt1 or to downstream Akt signaling substrates. The data are indicated as semiquantitative data, as mean ± SEM for the relative abundance of proteins.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Transgenic Assay, Expressing, Mutagenesis, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) Western blot analysis of MCF-7 cells transduced with an expression vector encoding cyclin D1 with antibodies as indicated to cyclin D1, Akt1, Akt1(Ser473), and the Akt1 signaling pathway and its substrate TSC2 (Ser939). The comparison of FLAG-tagged cyclin D1 (high molecular weight, labeled “Ex” for exogenous) and endogenous (labeled “End”) cyclin D1 indicates a 2-fold increase in cyclin D1 abundance. The data are representative of n = 3 separate experiments. (B and C) (B) Cyclin D1 −/− cells transduced with a retroviral vector for (B) cyclin D1 or (C) cyclin D1 WT and cyclin D1 KE with antibodies as indicated. (D and E) In (D), cyclin D1 immune-precipitation kinase assays were conducted using GST fusion proteins as substrates including pRB, and Akt1. Left panel: proteins on an SDS-PAGE stained with Coomassie. Right panel: γ 32 p IP-kinase assay reactions, with relative incorporation into substrates indicated in (E) as mean ± SEM for n = 3 separate experiments. (F) Representative mass spectrometry spectrum to map the Akt1 Ser473 phosphorylation status in vivo . The liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrum of the singly phosphorylated doubly charged peptide RPHFPQFpSYSASGTA representing S473 in the modified Akt1 sequence. The neutral loss of phosphate confirms the phosphorylation status, and sites are localized to S10 (S473 full length) in the peptide based on the b-ion series (N-terminal fragments) starting at b7 and the y-ion series starting at y10 that contain a phosphate group. (G) Cyclin D1 immune-precipitation kinase assays were conducted using GST fusion proteins including Akt1-WT and Akt1 Ser473 mutation. Left panel: proteins on an SDS-PAGE stained with Coomassie. Right panel: γ 32 P IP-kinase assay reactions.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Western Blot, Transduction, Expressing, Plasmid Preparation, Molecular Weight, Labeling, SDS Page, Staining, IP-Kinase Assay, Mass Spectrometry, In Vivo, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Modification, Sequencing, Mutagenesis

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A–F) The PLA for endogenous Akt1 with WT cyclin D1 or cyclin D1 mutants is indicated by red dots, and nucleus was stained by DAPI. Cyclin D1 expression plasmids were introduced by transduction of cyclin D1 −/− 3T3 cells. 3D reconstruction of the z stack is indicated for representative examples of multiplicate experiments. (G) Schematic representation of cyclin D1 expression plasmids used. (H) Model of the murine cyclin D1-CDK4/CDK6-Akt1 (C-terminal peptide) complex. The model indicates cyclin D1 and CDK4/CDK6 in blue and yellow cartoons, respectively. The Akt1 c-terminal peptide bound at the active site of CDK4/CDK6 is indicated in green stick. The ATP and Mg 2+ ions are indicated in blue sticks and red spheres, respectively.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Staining, Expressing, Transduction

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) The fluorescent intensity (FI) of LS456 in cyclin D1 −/− 3T3 cells transduced with either vector, cyclin D1 WT , or cyclin D1 KE . The FI of LS456 decreased in the 800-nm channel and increased in the 700-nm channel in response to insulin, indicated as fluorescent images in cells. (B) Quantitative analysis of the FI in cyclin D1 WT versus cyclin D1 KE or vector control. Fluorescent images in cells are superimposed on differential interference contrast images. Scale bars, 10 μm. Data are presented as mean ± SEM for n = 8 separate cells. (C) Schematic representation of c- fos promoter luciferase reporter genes including point mutations of the SRE and TCF site. (D and E) c- fos -LUC promoter activity in MCF-7 cells (D) transfected with expression vectors encoding either cyclin D1 WT or cyclin D1 KE or (E) transfected with c- fos -LUC reporter mutants. (F–H) Cyclin D1 −/− 3T3 cells were co-transfected with expression vector encoding c- fos -LUC WT or mutant and activated Akt (myr-Akt1) or (F) cyclin D1, (G) membrane-localized cyclin D1, or (H) nuclear-localized cyclin D1.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Transduction, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Mutagenesis

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: (A) Schematic representation of transgenic mice used for gene expression analysis. MMTV-Akt +/− mice were intercrossed with MMTV-ErbB2 transgenics, and the mammary epithelium was used for a source of mRNA. The cyclin D1-induced state was recapitulated through doxycycline induction of MMTV-RtTA-cyclin D1 transgenics treated for 10 days. (B) Pie diagrams representing the number of genes either induced or repressed by Akt or cyclin D1. (C) KEGG pathway analysis used to identify functional pathways regulated by either Akt1 or cyclin D1 and those regulated by both Akt1 and cyclin D1 . (D) The names of pathways identified by KEGG analysis regulated by both cyclin D1 and Akt1 in the mammary gland (migration 1, adherens junction; migration 2, tight junction). (E) Schematic representation of the relative fold enrichment in gene number of the individual KEGG pathways regulated by both cyclin D1 and Akt1.

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Transgenic Assay, Expressing, Functional Assay, Migration

Journal: Cell reports

Article Title: Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation

doi: 10.1016/j.celrep.2020.108151

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified Akt1 used in the cyclin D1 immune-precipitation kinase assays was from OriGene, (AKT1, CAT#TP320257) or GST fusion proteins for pRB, which contains 2 CDK phosphorylation sites , Akt1 or Akt1-S473A, were generated in this laboratory as previously described ( ).

Techniques: Recombinant, In Situ, shRNA, Plasmid Preparation, Mutagenesis, Software