Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: miR-214 inhibition and ATF4 overexpression reverse MEG3 knockdown-mediated decreases in FoxO1, G6pc and Pepck protein expression. Cells were transfected with the indicated plasmids, followed by treatment with 0.5 mM palmitate for 4 h. (A–D) Western blot analysis revealed that MEG3 knockdown significantly suppressed the palmitate-mediated increase in (B) FoxO1, (C) Pepck, and (D) G6pc protein expression. (E–H) By contrast, the miR-214 inhibitor additionally induced the palmitate-mediated increase in (F) FoxO1, (G) Pepck and (H) G6pc protein expression. (I–L) ATF4 overexpression also induced the palmitate-mediated increase in (J) FoxO1, (K) Pepck and (L) G6pc protein expression. β-tubulin served as the loading control in the western blot analyses. * P<0.05 vs . group 1; # P<0.05 vs. group 3; $ P<0.05 vs . group 4. si, small interfering RNA; ctrl, control; MEG3, maternally expressed gene 3; miR, microRNA; FoxO1, forkhead box protein O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit; ATF4, activating transcription factor 4.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Inhibition, Over Expression, Knockdown, Expressing, Transfection, Western Blot, Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: miR-214 inhibition and ATF4 overexpression reverse the MEG3 knockdown-mediated decrease in FoxO1, G6pc and Pepck mRNA expression. Cells were transfected with the indicated plasmids, followed by treatment with 0.5 mM palmitate for 4 h. (A–C) RT-qPCR results revealed that MEG3 knockdown significantly suppressed the palmitate-mediated increase in (A) FoxO1, (B) Pepck, and (C) G6pc mRNA expression. (D–F) By contrast, the miR-214 inhibitor induced the palmitate-mediated increase in (D) FoxO1, (E) Pepck, and (F) G6pc mRNA expression. (G–I) ATF4 overexpression also induced the palmitate-mediated increase in (G) FoxO1, (H) Pepck, and (I) G6pc mRNA expression. * P<0.05 vs . group 1; # P<0.05 vs. group 3; $ P<0.05 vs . group 4. miR, microRNA; ATF, activating transcription factor 4; MEG3, maternally expressed gene 3; FoxO1, forkhead box protein O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit; si, small interfering RNA; ctrl, control; MEG3.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Inhibition, Over Expression, Knockdown, Expressing, Transfection, Quantitative RT-PCR, Small Interfering RNA, Control

Journal: International Journal of Molecular Medicine

Article Title: lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression

doi: 10.3892/ijmm.2018.3975

Figure Lengend Snippet: Proposed associations between target genes. Palmitate time-dependently increased MEG3 and ATF4 expression, but decreased miR-214 expression. MEG3 functioned as a competing endogenous RNA of miR-214 to upregulate ATF4 expression, leading to the promotion of FoxO1 and its downstream gluconeogenic enzymes G6pc and Pepck. This thereby increases gluconeogenesis and promotes insulin resistance. MEG3, maternally expressed gene 3; miR, microRNA; ATF4, activating transcription factor 4; FoxO1, Forkhead box protein O1; O1; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase catalytic subunit.

Article Snippet: The PVDF membranes were then blocked with 5% fat-free milk for 1 h at room temperature and sequentially incubated at 4°C overnight with the corresponding primary antibodies against ATF4 (1:1,000; cat. no. ab23760; Abcam, Cambridge, MA, USA), FoxO1 (1:1,000; cat. no. ab39670; Abcam), Pepck (1:200; cat. no. sc-377027; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and G6pc (1:1,000; cat. no. TA334651; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 is a potent PI3Kγ/PKA disruptor peptide. A , chemical structure of DRI-Pep #20. The amino acid sequence of DRI-Pep #20 comprises the nonnatural D-peptide RHQGK, the D-retroinverso (DRI)-isoform of the cell penetrating peptide Penetratin 1 (P1) and a glycine (G) linker. B , schematic representation of the fluorescence spectroscopy assays for the characterization of the interaction between DRI-Pep #20 (or PI3Kγ MP) and the recombinant fluorescein 5-maleimide–labeled PKA-RIIα (PKA-F5M). C , steady-state emission spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 (0–20 μM). K D : dissociation constant. Inset, nonlinear fitting of the fluorescence intensity maxima obtained at various concentrations of DRI-Pep #20 for the monitoring of bio-labeled PKA. K A : association constant. D , for kinetic analysis, fluorescence spectra of PKA-F5M in the presence of increasing concentrations of DRI-Pep #20 or PI3Kγ MP (inset) were analyzed and fitted to a single exponential function to obtain the observed rate constant ( k obs ). The binding of DRI-Pep #20 or PI3Kγ MP to biolabeled PKA was investigated under pseudo -first-order conditions, and the kinetic constants, k on and k off , were determined. E , schematic representation of the displacement assay between DRI-Pep #20 (or PI3Kγ MP) and the PI3Kγ/PKA-F5M complex. F , percentage displacement of the PI3Kγ/PKA-RIIα complex by DRI-Pep #20 or PI3Kγ MP, calculated from steady-state emission spectra of the PI3Kγ/PKA-F5M complex in the presence of increasing concentrations of the peptides (0–5 μM). The displacement efficiency was expressed as percentage of the binding between PI3Kγ and PKA-F5M relative to that in the absence of peptides. G , cAMP concentrations in peritoneal macrophages from WT (in green ) and PI3Kγ −/− mice (in gray ) treated with DRI-Pep #20 (1–25 μM) for 30 min. The amount of cAMP was expressed as percentage of cAMP accumulation observed in untreated PI3Kγ −/− cells. n ≥ 6 technical replicates from N > 3 independent experiments. ∗∗∗ p < 0.001 WT versus PI3Kγ −/− and # p < 0.05, ## p < 0.01, and ### p < 0.001 UT versus DRI-Pep #20 by one-way ANOVA, followed by Bonferroni’s post hoc test. Data are means ± SD. AU, arbitrary units; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα; PI3Kγ, phosphoinositide 3-kinase gamma; PKA-F5M, fluorescein 5-maleimide–labeled PKA-RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Sequencing, Fluorescence, Spectroscopy, Recombinant, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Binding kinetics of the interaction between DRI-Pep #20 or PI3Kγ MP and PKA-RIIα

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the binding between DRI-Pep #20 and PKA-RIIα. A , DRI-Pep #20 structure prediction by PEP-FOLD3.5. P1-G and RHQGK domains are shown as cartoons in gray and red , respectively. R-1, H-2, Q-3, and K-5 residues are indicated and shown as sticks . B , circular dichroism spectra of DRI-Pep #20 showing a peak at 190–240 nm. The percentage of α-helical and β-sheet secondary structures calculated by the K2D3 software are indicated. C , molecular docking simulation of the interaction between DRI-Pep #20 and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of DRI-Pep #20 in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. The key residues involved in the binding are indicated and shown as sticks , with DRI-Pep #20 residues in bold . Hydrogen bonds between DRI-Pep #20 and PKA-RIIα are indicated by yellow dashed lines . In ( A and C ), the structural models were developed using PyMOL. DRI, D-retroinverso; HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Structural Proteomics, Binding Assay, Circular Dichroism, Software

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: Structural prediction of the native binding between the N-terminal domain of PI3Kγ and PKA-RIIα. A , molecular docking simulation of the interaction between PI3Kγ and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of residues 109 to 159 of PI3Kγ in complex with residues 2 to 44 of the PKA-RIIα dimer ( green cartoon) is shown. The amino acids critical for the binding between the two proteins are shown and indicated as sticks , with the residues of PI3Kγ in bold . The putative PKA-binding motif of PI3Kγ (126–150) is shown in orange and blue . The sequence in orange indicates the region of PI3Kγ that was identified as being at the core of the interaction (KATHR). Hydrogen bonds between PI3Kγ and PKA-RIIα are indicated by yellow dashed lines . B , structural prediction of the KATHR sequence by PEP-FOLD3.5. KATHR and P1-G domains are shown as cartoons in orange and gray , respectively. K-18, H-21 and R-22 residues of the KATHR sequence (corresponding to K-126, H-129 and R-130 of native PI3Kγ) are indicated and shown as sticks . C , molecular docking simulation of the interaction between KATHR and the PKA-RIIα dimer by HADDOCK 2.4. The docked pose of KATHR in complex with residues 2 to 44 of PKA-RIIα (cartoon in green ) is shown. Yellow dashed lines indicate hydrogen bonds between KATHR and 2 to 44 PKA-RIIα. The amino acids critical for the binding are indicated and shown as sticks , with KATHR residues in bold . Throughout, the structural models were developed using PyMOL. HADDOCK, high ambiguity driven biomolecular DOCKing; PI3Kγ, phosphoinositide 3-kinase gamma; PKA, protein kinase A; PKA-RIIα, PKA regulatory subunit RIIα.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: Structural Proteomics, Binding Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: A nonnatural peptide targeting the A-kinase anchoring function of PI3Kγ for therapeutic cAMP modulation in pulmonary cells

doi: 10.1016/j.jbc.2024.107873

Figure Lengend Snippet: DRI-Pep #20 increases cAMP levels locally in vivo in the airway tract of mice. A , schematic representation of the treatment schedule. Mice received DRI-Pep #20 through intratracheal (i.t.) instillation. B – D , cAMP concentrations in tracheas ( B ), lungs ( C ) and hearts ( D ) from BALB/c mice 24 h after i.t. instillation of different doses of DRI-Pep #20 (0–750 mg/kg). Values in brackets indicate the dose of DRI-Pep #20 expressed as mg/kg. The number of mice (n) ranged from three to six per group. EC 50 , median effective concentration. E – G , cAMP concentrations in tracheas ( E ), lungs ( F ) and hearts ( G ) from WT and PI3Kγ −/− mice 24 h after i.t. instillation of 10 μg/Kg DRI-Pep #20 (in green ) or PBS (in gray ). The number of mice (n) ranged from three to four per group. In ( A and B ), ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 by one-way ANOVA, followed by Bonferroni’s post hoc test. In ( E and F ) ∗ p < 0.05 and ∗∗ p < 0.01 PBS versus DRI-Pep #20 by two-way ANOVA test, followed by Bonferroni’s post hoc analysis. Throughout, data are means ± SD. DRI, D-retroinverso; PI3Kγ, phosphoinositide 3-kinase γ.

Article Snippet: Recombinant human PKA regulatory subunit RIIα (PKA-RIIα; product code: PK-PKA-R2A025) and catalytic subunit Cα (PKA-Cα; product code: PK-PKA-HCA050) were purchased from Biaffin GmbH & Co KG. PI3Kγ catalytic subunit (p110γ) was from Origene Technologies (TP307790).

Techniques: In Vivo, Concentration Assay

Journal: Molecular Cancer Research

Article Title: AKT1 E17K Inhibits Cancer Cell Migration by Abrogating β-Catenin Signaling

doi: 10.1158/1541-7786.mcr-20-0623

Figure Lengend Snippet: Figure 4. AKT2, but not AKT1, increases the transcriptional activity of b-catenin on the ZEB1 promoter, and decreases E-cadherin expression. A, p53ko/E17K cells were infected with a CRISPR/Cas9 lentivirus targeting AKT1 and p53ko cells were infected with a CRISPR/Cas9 lentivirus targeting AKT2. Cells lysates were then subjected to ChIP using a b-catenin antibody. Top: Western blot of whole cell lysates from the indicated p53ko/E17K cells and p53ko cells before and after AKT1 knockdown (AKT1KD) or AKT2 knockdown (AKT2KD) as specified. Bottom: Relative fold of ChIP pull-down of the ZEB1 promoter (b-catenin fold enrichment) was quantified by real-time qPCR, normalized by both input DNA and IgG values. Error bars, mean SEM (n ¼ 3). P values were determined by one-way ANOVA followed by Tukey post hoc test. , P < 0.0001. B, Western blots of lysates from MCF-10A p53ko/E17K or MCF7 PIK3CA WT cells expressing HA-tagged Myr-AKT2 and FLAG-tagged Myr-AKT1 alone and in combination. C, Western blot of lysates from p53ko cells: Left: infected with PTEN CRISPR/Cas9 lentivirus; Right: infected with PIK3CA E545K lentivirus. Cell lysate of parental MCF-10A cells was used as an E-cadherin–positive control.

Article Snippet: PIK3CA E545K–mutant cDNA (Cat#RC400348), purchased from OriGene, was inserted into the AsiS I and Mlu I sites of plasmid Lenti-C-Myc-DDK-IRES-Neo (Cat#PS100081, OriGene), to generate PIK3CA-E545K lentiviral expression vector.

Techniques: Activity Assay, Expressing, Infection, CRISPR, Western Blot, Knockdown, Positive Control

Journal: Cancer Research

Article Title: Phosphorylation of Carbonic Anhydrase IX Controls Its Ability to Mediate Extracellular Acidification in Hypoxic Tumors

doi: 10.1158/0008-5472.can-11-2520

Figure Lengend Snippet: Figure 3. Phosphorylation and dephosphorylation of Thr443. A, in vitro kinase assay was carried out with the CS-PKA on GST fusion proteins containing the IC domain of CA IX in the wt version and in the mutated version with Thr443 replaced by Ala (T443 ! A). The upper part of the figure represents a CBB-stained gel (loading control), the lower part represents autoradiograph of the proteins phosphorylated in the presence of [g-32P]ATP. B, in vitro phosphatase assay was carried out with heterodimeric and heterotrimeric forms of PP2A on PKA-phosphorylated GST-tagged wt-IC fragment of CA IX. The upper part of the figure represents a gel with CBB-stained substrate proteins (loading control), the lower part represents autoradiograph. The data show that Thr443 is a substrate residue for PKA and PP2A. The experiments (in A and B) were repeated twice and representative results are shown. C, CA IX was immunoprecipitated by the M75 MAb from cell extracts, in vitro phosphorylated by CS-PKA, subjected to PAGE, blotted and incubated with the P-T443 CA IX antibody. In vitro phosphorylated GST-tagged wt-IC domain of CA IX protein (GST-IC) was used as a positive control. D, all 3 blots contain PAGE-resolved extracts from HEK293T/17 cells transfected with different combinations of the following plasmids: pcDNA3.1 (p1), pSG5C (p2), pSG5C-MN/CA9 (pCA9), pcDNA3.1- CSPKA (pCa), pcDNA3.1dnPKA (pdn). The plasmids were cotransfected in a ratio of 10:1. The blots were incubated with antibodies as indicated on the left side. The data show that phosphorylation of CA IX, activation of PKA and phosphorylation of PKA substrates occur only in the presence of CS-PKA. The experiment was repeated 4 times.

Article Snippet: HEK293T/17 cells were cotransfectedwith various combinations of the following plasmids: pCalpha-EV-PKA encoding CSPKA (pCa, catalytic subunit C alpha, 15,310 Addgene plasmid; ref. 17), pcDNA3.1þdnPKA encoding DN-PKA (pdn), pSG5CMN/CA9 encoding CA IX (pCA9) and respective empty plasmidsbyNeoFectin in the ratio10:1, 4mgofplasmidDNA in total.

Techniques: Phospho-proteomics, De-Phosphorylation Assay, In Vitro, Kinase Assay, Staining, Control, Autoradiography, Phosphatase Assay, Residue, Immunoprecipitation, Incubation, Positive Control, Transfection, Activation Assay

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Overexpression of Akt isoforms and DNA-PKcs in A549 or HEK293T cells. Expression of Akt and DNA-PKcs fusion proteins. ( a ) Schematic overview of AKT1, AKT2 and AKT3 constructs fused to mCherry as well as the constructs expressing different subdomains of DNA-PKs fused to eGFP. ( b ) A549 cells were transfected with the indicated constructs. Twenty-four hours after the transfection, fluorescent images were acquired. Cells expressing mCherry or eGFP alone served as controls. Scale bars: 20 μm. ( c ) HEK293T cells were transfected with the indicated constructs. Twenty-four hours after the transfection, the cells were lysed and eGFP fusion proteins were precipitated using the GFP-Trap. Whole-cell lysates (mCherry) and the bound fractions (eGFP) were subjected to SDS-PAGE followed by western blot analysis using antibodies specific for mCherry or eGFP.

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Over Expression, Expressing, Construct, Transfection, SDS Page, Western Blot

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Akt1 and Akt3 but not Ak2 interact with DNA-PKcs. K-RAS-mutated A549 cells were transfected with the indicated plasmids. Forty-eight hours after the transfection, cells were irradiated with 4 Gy and lysed 10 min post-irradiation. IP of eGFP was performed using GFP-Trap. The co-IPs for Akt1 ( a ) Akt2 ( b ) and Akt3 ( c ) were analyzed by western blotting using Akt-specific antibodies. The densitometry values represent the ratios of Akt1/eGFP ( a ), Akt2/eGFP ( b ) and Akt3/eGFP ( c ). Cells were transfected with eGFP-DNA-PKcs-N and were either mock irradiated or irradiated with 4 Gy. Subsequently, the cells were lysed at the indicated times post-irradiation and the eGFP-tagged proteins were precipitated from the soluble protein fraction. The bound fractions were subjected to SDS-PAGE and immunoblot analysis using anti-eGFP ( d , e ), antibodies against the Akt1 and Ak2 isoforms ( d ) or against the Akt2 and Akt3 isoforms ( e ). Ponseau staining was shown for locating protein bands on immunoblots.

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Transfection, Irradiation, Western Blot, SDS Page, Staining

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Targeting Akt inhibits Akt/DNA-PKcs complex formation. K-RAS-mutated A549 cells were transfected with eGFP-PKcs (a.a. 1-421) and mCherry-Akt1. ( a ) Forty-eight hours after transfection, the cells were treated with DMSO (D) or 10 μ M of MK2206 (MK) for 1 h and then irradiated with 4 Gy. The cells were lysed 10 min post-IR and eGFP-DNA-PKcs-N was precipitated as described. The input and bound fractions were subjected to SDS-PAGE and immunoblot analysis, and eGFP was detected in the inputs and under the IP conditions as the loading control. The experiment was performed in two biological replicates. The results from one experiment are shown. ( b ) The cells were lysed 10 min past-IR and immunoblot analysis was subjected. The phosphorylation of Akt (Ser-473) and PRAS40 (Thr-246) was analyzed by immunoblotting in the whole-cell lysates using phospho-specific antibodies. The blots were stripped and re-probed with the antibodies against Akt1 and PRAS40. Data indicates mean P-Akt (Ser-473)±S.E.M. from three independent experiments. The asterisks indicate a significant inhibition of Akt phosphorylation following pretreatment with MK2206 (5 μ M) for 2 h followed by irradiation with 4 Gy (* P <0.05, *** P <0.001, Student's t -test).

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Transfection, Irradiation, SDS Page, Western Blot, Inhibition

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Effect of Akt isoforms and DNA-PKcs on post-irradiation cell survival of K-RAS-mutated A549 cells. ( a ) Forty-eight hours after the transfections with the indicated siRNAs, the cells were plated in six-well plates, the colonies were stained after about 10 days and the plating efficiencies were calculated by dividing the number of colonies formed to the number of cells seeded. The data presented are the mean plating efficiencies (PE)±S.E.M. of 12 replicates from two independent experiments. ( b ) Transfected cells with indicated siRNA were plated and X-ray irradiated 24 h later and then incubated for 10 days. Thereafter, the colonies were stained, and the survival fractions (SF) were calculated as described in the Materials and Methods section. The data presented are the mean survival fraction±S.E.M. of 12 replicates from two independent experiments. ( c ) Confluent A549 cells were treated with the vehicle (DMSO) or the DNA-PKcs inhibitor NU7026 at indicated concentrations for 1 h and then irradiated with 4 Gy. Protein samples were isolated 30 min after irradiation, and levels of P-DNA-PKcs (Ser-2056) and P-DNA-PKcs (Thr-2609) were determined by immunoblotting. The blots were then stripped and incubated with the DNA-PKcs antibody. ( d ) A549 cells were plated in six-well plates and 24 h later were treated with the vehicle (DMSO) or the indicated concentrations of the DNA-PKcs inhibitor NU7026 for 1 h. The cultures were then irradiated and incubated for 10 days. Thereafter, the colonies were stained, and the clonogenic fractions were calculated as described in Materials and Methods section. The data presented are the mean survival fraction±S.E.M. of six replicates from the parallel experiments. The asterisks indicate a statistically significant inhibition of plating efficiency ( a ) and radiosensitization after knockdown of Akt1 or Akt3 ( b ) (* P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: Irradiation, Transfection, Staining, Incubation, Isolation, Western Blot, Inhibition

Journal: Cell Death Discovery

Article Title: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

doi: 10.1038/cddiscovery.2017.72

Figure Lengend Snippet: Knockdown of Akt1 and Akt3 but not Akt2 inhibits proliferation and tumor growth in K-RAS-mutated MDA-MB-231 cells. ( a ) Cells (3×10 4 ) were plated in 6 cm culture dishes. At the indicated days after seeding, cells were counted and graphed. The data points represent the mean cell counts±S.E.M. of eight parallel experiments from two independent experiments. Asterisks indicate significant prolongation of PDT after knockdown of Akt1 and Akt3 compared with scramble-shRNA (scr-shRNA) (* P <0.05, *** P <0.001). ( b ) Protein samples were isolated from the cells counted on day 7 and expression of Akt isoforms was tested by immunoblotting. ( c ) Indicated cells (3×10 4 ) were plated for 24 h and treated with DNA-PKcs inhibitor NU7441 (10 μ M). Cells were count on day 6 after treatment and graphed. Data present mean cells numbers of eight data±S.E.M. obtained from two independent experiments. ( d ) Nude mice were injected with indicates cells (2×10 6 cells) in both dorsal flank and tumor growth assay was performed as described in Materials and Methods section. Data present mean tumor volume±S.E.M. of 14 tumors (seven mice) inoculated with MDA-MB-231-expressing scr-shRNA and of 12 tumors from six animals inoculated with MDA-MB-231 cells expressing Akt1-, Akt2- or Akt3-shRNA. Asterisks indicate a significant tumor growth delay by knockdown of Akt1 as well as Akt3 (*** P <0.001) and increased in tumor volume by knockdown of Akt2 (* P <0.05), measured 6 weeks after inoculation. ( e ) Representative images of tumors following inoculation of MDA-MB-231 cells expressing scr-shRNA as well as shRNA against the Akt isoforms.

Article Snippet: Prior to western blot analysis, the eGFP-tagged DNA-PKcs domains were enriched by IP with GFP-Trap (ChromoTek GmbH) according to the manufacturer’s protocol.

Techniques: shRNA, Isolation, Expressing, Western Blot, Injection, Growth Assay