inactive p70s6k recombinant protein  (SignalChem)

Journal: Frontiers in Oncology

Article Title: Ethyl Ferulate Suppresses Esophageal Squamous Cell Carcinoma Tumor Growth Through Inhibiting the mTOR Signaling Pathway

doi: 10.3389/fonc.2021.780011

Figure Lengend Snippet: Ethyl ferulate strongly inhibits the mammalian target of rapamycin (mTOR) signaling pathway. (A) The effect of ethyl ferulate on epidermal growth factor (EGF)-induced kinase signaling in JB6 cells. Serum-starved [without fetal bovine serum (FBS); 24 h] cells were treated with different concentrations of ethyl ferulate for 6 h followed by treatment with EGF for 30 min. (B) The effect of ethyl ferulate on various kinase signaling proteins in esophageal squamous cell carcinoma (ESCC) cells. Cells were treated with ethyl ferulate for 24 h, and then various signaling proteins were examined by Western blotting. (C) Ethyl ferulate directly binds to recombinant mTOR protein and mTOR present in KYSE510 cell lysates. The recombinant mTOR protein or cell lysate was incubated with Sepharose 4B beads or ethyl ferulate-conjugated Sepharose 4B beads. Pulled down proteins were examined by Western blotting. (D) The effect of ethyl ferulate on mTOR kinase activity. mTOR kinase activity was assessed by in vitro kinase assay using active mTOR and inactive p70S6K proteins. For all experiments, similar results were shown as mean values ± SD for 3 independent experiments and band density is shown as a bar graph. One-way analysis of variance (ANOVA) was used to analyze the data. The asterisk (*) indicates a significant ( P < 0.05) inhibitory effect of ethyl ferulate.

Article Snippet: Active mTOR recombinant protein used for in vitro kinase assay or in vitro pull-down assay was purchased from Thermo Fisher (Shanghai, China), and inactive p70S6K recombinant protein used for in vitro kinase assay was purchased from SignalChem (Richmond, BC, Canada).

Techniques: Western Blot, Recombinant, Incubation, Activity Assay, In Vitro, Kinase Assay

Journal: Frontiers in Oncology

Article Title: Ethyl Ferulate Suppresses Esophageal Squamous Cell Carcinoma Tumor Growth Through Inhibiting the mTOR Signaling Pathway

doi: 10.3389/fonc.2021.780011

Figure Lengend Snippet: Ethyl ferulate reduces esophageal squamous cell carcinoma (ESCC) patient-derived xenograft tumor growth in vivo. Mice were divided into two groups to assess the effect of ethyl ferulate on ESCC patient-derived xenograft (PDX) tumor growth. Groups are as follows: 1) vehicle group or 2) group treated with 100 mg/kg of ethyl ferulate. Tumor-bearing mice were orally treated (by gavage) with ethyl ferulate or vehicle for 32 (LEG110; n = 10) or 53 (LEG45; n = 7) days. Tumor volumes were measured on the days indicated. The effect of ethyl ferulate on ESCC tumor growth in (A) LEG110 or (B) LEG45 ESCC PDX tissues. For panels (A, B) , data are shown as mean ± SE from each group. The effect of ethyl ferulate on Ki-67 expression in (C) LEG110 or (D) LEG45 ESCC PDX tissues (n = 5). Three slices per PDX tissue were analyzed. Treated or untreated groups of tumor tissues were stained with antibodies to detect Ki-67 (×100; scale bar: 100 μm; ×20; scale bar: 50 μm). (E, F) Ethyl ferulate inhibits phosphorylated mammalian target of rapamycin (mTOR), p70S6K, and AKT protein expression in esophageal tumor tissues. Tumor tissues from each group were immunoblotted with antibodies to detect mTOR, p70S6K, and AKT and phosphorylated mTOR, p70S6K, AKT, and β-actin. β-Actin was used to verify equal protein loading. Band density was measured using the ImageJ (NIH) software program, and the results are shown as a bar graph. One-way ANOVA was used to compare significant differences. The asterisk (*) indicates a significant (* P < 0.05; ** P < 0.01) inhibitory effect of ethyl ferulate treatment.

Article Snippet: Active mTOR recombinant protein used for in vitro kinase assay or in vitro pull-down assay was purchased from Thermo Fisher (Shanghai, China), and inactive p70S6K recombinant protein used for in vitro kinase assay was purchased from SignalChem (Richmond, BC, Canada).

Techniques: Derivative Assay, In Vivo, Expressing, Staining, Software

Journal: Frontiers in Oncology

Article Title: Ipriflavone Suppresses Growth of Esophageal Squamous Cell Carcinoma Through Inhibiting mTOR In Vitro and In Vivo

doi: 10.3389/fonc.2021.648809

Figure Lengend Snippet: Ipriflavone is a novel mTOR inhibitor. (A) Effect of Ipriflavone on EGF‐induced kinase signaling molecules in JB6 cells. After serum starvation for 24 h, cells were treated with different doses of Ipriflavone for 6 h followed by EGF treatment for 30 min. various signaling molecules were analyzed by Western blotting. (B) Effect of Ipriflavone on various signaling molecules in KYSE450 ESCC cells. Cells were treated with Ipriflavone for 24 h and signaling molecule proteins were examined by Western blotting. (C) Effect of Ipriflavone on mTOR kinase activity was assessed by an in vitro kinase assay using active mTOR and inactive p70S6K proteins. The activity of mTOR was determined by Western blotting using a phosphorylated p70S6K antibody. AZD8055 (mTOR inhibitor) was used as a positive control. For all data, similar results were observed from three independent experiments and band density was measured using the Image J (NIH) software program. The asterisk (*) indicates a significant ( p < 0.05) difference. (D) Modeling of Ipriflavone binding with mTOR. ATPγS ( D , upper left panel ) and Ipriflavone ( D , upper right panel ) binding with mTOR at the ATP binding pocket. ( D , lower panel ) Ligand Interaction Diagram (LID) of the binding. The Ipriflavone is shown as stick. LID legend is shown below.

Article Snippet: The inactive p70S6K recombinant protein (100 ng, Signalchem) and ATP (Cell Signaling Technology) were added and the mixtures were incubated at 30°C for 30 min.

Techniques: Western Blot, Activity Assay, In Vitro, Kinase Assay, Positive Control, Software, Binding Assay