Journal: International Journal of Molecular Sciences

Article Title: Synthesis, Molecular Docking, In Vitro and In Vivo Studies of Novel Dimorpholinoquinazoline-Based Potential Inhibitors of PI3K/Akt/mTOR Pathway

doi: 10.3390/ijms231810854

Figure Lengend Snippet: FDA-approved inhibitors of PI3K/Akt/mTOR pathway [ , , ].

Article Snippet: The PI 3-Kinase (Class I) HTRF Assay Kit (Cat# 33-016 from Millipore) was used to determine the activity of recombinant PI3K (p110α/p85α) human (Cat# 14-602 from Millipore).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Synthesis, Molecular Docking, In Vitro and In Vivo Studies of Novel Dimorpholinoquinazoline-Based Potential Inhibitors of PI3K/Akt/mTOR Pathway

doi: 10.3390/ijms231810854

Figure Lengend Snippet: Compound 7c suppresses the PI3K/Akt/mTOR signaling pathway. ( a – i ) MCF7 cells were treated with different concentrations of 7c or 500 nM wortmannin (W) for 24 h. Cell lysates were immunoblotted with antibodies against PI3K/Akt/mTOR-related total or phosphorylated (p) proteins ( a – f ) and PARP1/cleaved PARP1 ( g , h ). ( i ) Control α-tubulin. ( j , k ) The ratios of p-proteins to total ones for compound 7c (circles) or wortmannin (triangles) ( j ) and cleaved PARP1 to full PARP1 ( k ) as measured by ImageJ program from two independent experiments.

Article Snippet: The PI 3-Kinase (Class I) HTRF Assay Kit (Cat# 33-016 from Millipore) was used to determine the activity of recombinant PI3K (p110α/p85α) human (Cat# 14-602 from Millipore).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Synthesis, Molecular Docking, In Vitro and In Vivo Studies of Novel Dimorpholinoquinazoline-Based Potential Inhibitors of PI3K/Akt/mTOR Pathway

doi: 10.3390/ijms231810854

Figure Lengend Snippet: Inhibition of PI3Kα enzymatic activity with compounds 7b (blue) or 7c (red).

Article Snippet: The PI 3-Kinase (Class I) HTRF Assay Kit (Cat# 33-016 from Millipore) was used to determine the activity of recombinant PI3K (p110α/p85α) human (Cat# 14-602 from Millipore).

Techniques: Inhibition, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Synthesis, Molecular Docking, In Vitro and In Vivo Studies of Novel Dimorpholinoquinazoline-Based Potential Inhibitors of PI3K/Akt/mTOR Pathway

doi: 10.3390/ijms231810854

Figure Lengend Snippet: Docking of the compounds 7b ( a , b ) and 7c ( c , d ) into the catalytic site of PI3Kα ( a , c ) and PI3Kγ ( b , d ). The gray dotted line is hydrophobic interactions, the blue solid line is hydrogen bonds, the light green dotted line is parallel π-π stacking, dark green dashed—normal π-π stacking. yellow dotted line—salt bridge. Pictures 5E and 5F represent a superposition of 7b , 7c and Gedatolisib 8 in the catalytic site of PI3Kα ( e ) and PI3Kγ ( f ). Gedatolisib ( 8 ) is colored in blue, 7b —red and 7c —green. Visualization of the docking results (protein-ligand complexes) was carried out using the PLIP service. The superposition of ligands was visualized via UCSF Chimera program.

Article Snippet: The PI 3-Kinase (Class I) HTRF Assay Kit (Cat# 33-016 from Millipore) was used to determine the activity of recombinant PI3K (p110α/p85α) human (Cat# 14-602 from Millipore).

Techniques:

Journal: Neoplasia (New York, N.Y.)

Article Title: A highly selective PI3Kδ inhibitor BGB-10188 shows superior preclinical anti-tumor activities and decreased on-target side effects on colon

doi: 10.1016/j.neo.2024.101053

Figure Lengend Snippet: BGB-10188 showed potent BCR downstream PI3Kδ/AKT signaling inhibition in normal mouse B cells. BALB/c mice were treated with 0, 0.3, 3, 10 and 30 mg/kg of BGB-10188 once and euthanized at different time points after dosing as indicated. Whole blood was collected for both pAKT detection and BGB-10188 concentration measurement. (A) pAKT Inhibition of BGB-10188 in mouse peripheral blood B cells. Whole blood was stimulated by anti-mouse IgD antibody for 7 min at 37 °C for activating B-cell receptors, thus inducing the phosphorylation of AKT. pAKT level in mouse peripheral blood B cells (IgD + B220 + cells) was detected by flow cytometry. (B) The plasma concentration of BGB-10188 measured by LC-MS/MS.

Article Snippet: PI3K p110δ/p85α (Promega, Cat#: V1771), p110α/p85α (Promega, Cat#: V1721), p110β/p85α (Promega, Cat#: V1751) and p110γ (ThermoFisher Scientific, Cat#: PV4786) kinase activity inhibition by BGB-10188 (BeiGene (Beijing) Co. Ltd) and idelalisib (Shanghai Scochem Tecknology) were tested in ADP-Glo TM Kinase Assay.

Techniques: Inhibition, Concentration Assay, Phospho-proteomics, Flow Cytometry, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Neoplasia (New York, N.Y.)

Article Title: A highly selective PI3Kδ inhibitor BGB-10188 shows superior preclinical anti-tumor activities and decreased on-target side effects on colon

doi: 10.1016/j.neo.2024.101053

Figure Lengend Snippet: BGB-10188 and zanubrutinib synergized in DLBCL TMD8 cell line and MCL MINO cell line. (A, C) Inhibition of TMD8 (A) and MINO (C) cell proliferation by co-treatment with BGB-10188 and zanubrutinib at a series of corresponding concentrations in a 6-day viability assay. (B, D) The synergy score analyzing the synergistic effect of BGB-10188 and Zanubrutinib in TMD8 (B) and MINO (D) cells. The synergy score is calculated by Loewe model and the interaction between two drugs is likely to be additive or synergistic when synergy score is from -10 to 10 or larger than 10. (E, F) Western Blot analysis of PI3Kδ and BTK downstream signaling. TMD8 (E) and MINO (F) cells were seeded into 6-well plates at a density of 1 × 10 6 per well in full RPMI-1640 medium. Cells were co-treated with 100 nM BGB-10188 and 10 nM zanubrutinib for 4 or 24 h respectively, dimethyl sulfoxide alone was added to the negative control cells.

Article Snippet: PI3K p110δ/p85α (Promega, Cat#: V1771), p110α/p85α (Promega, Cat#: V1721), p110β/p85α (Promega, Cat#: V1751) and p110γ (ThermoFisher Scientific, Cat#: PV4786) kinase activity inhibition by BGB-10188 (BeiGene (Beijing) Co. Ltd) and idelalisib (Shanghai Scochem Tecknology) were tested in ADP-Glo TM Kinase Assay.

Techniques: Inhibition, Viability Assay, Western Blot, Negative Control

Journal: Neoplasia (New York, N.Y.)

Article Title: A highly selective PI3Kδ inhibitor BGB-10188 shows superior preclinical anti-tumor activities and decreased on-target side effects on colon

doi: 10.1016/j.neo.2024.101053

Figure Lengend Snippet: BGB-10188 showed inhibition in both Treg percentage and Treg function in mouse blood but not in colon tissues where idelalisib does. (A–E) PI3Kδ deficiency or inhibition by BGB-10188 led to decreased Treg proliferation and tumor growth inhibition. (A–C) Effects of PI3Kδ deficiency on Treg and T cell proliferation. Splenocytes from P110δ WT/WT , P110δ D910A/WT , P110δ D910A/D910A mice ( n = 12, 6 female plus 6 male) were stained with CFSE (Cat:65-0850-84, Life Technologies) and activated by soluble 10 µg/ml anti-CD3 (145-2C11, Biolegend), 1 µg/ml anti-CD28 (16-0281-86, eBioscience™) and 500 IU/mL IL-2 in the u-bottom 96-well plates at 37°C for 3 days. Cell percentage with low CFSE fluorescence of Treg (A, CD4 + FoxP3 + population), non-Treg CD4 + cells (B, CD4 + FoxP3 - ) and CD8 + cells (C) detected by FACS were indicated as the proliferation rate. (D-E) Efficacy (D) and tumor Treg inhibition (E) of BGB-10188 in A20 model. A20 tumor cells (2.5 × 10 6 /mouse) were implanted subcutaneously in female BALB/c mice. On the next day after tumor inoculation, animals were randomly assigned according to the body weight and treated BID with vehicle (0.5 % MC) and 30 mg/kg or 60 mg/kg BGB-10188 for 22 days, n = 12. At the end of the study, mice were sacrificed to collect tumor and isolate the tumor infiltrated lymphocytes for Treg detection by flow cytometry. Log transformed tumor volume on the last day or Treg cell (Foxp3 + ) percentages between groups were analyzed by one-way ANOVA, * P < 0.05 was considered statistically significant. (F–K) BALB/c mice were orally treated with 30 or 60 mg/kg BGB-10188, and 60 or 100 mg/kg idelalisib. The mice were sacrificed after 6 days’ drug administration to collect blood (F–H) and colon (I–K) for immune cell profiling by flow cytometry.

Article Snippet: PI3K p110δ/p85α (Promega, Cat#: V1771), p110α/p85α (Promega, Cat#: V1721), p110β/p85α (Promega, Cat#: V1751) and p110γ (ThermoFisher Scientific, Cat#: PV4786) kinase activity inhibition by BGB-10188 (BeiGene (Beijing) Co. Ltd) and idelalisib (Shanghai Scochem Tecknology) were tested in ADP-Glo TM Kinase Assay.

Techniques: Inhibition, Staining, Fluorescence, Flow Cytometry, Transformation Assay

Journal: Neural Regeneration Research

Article Title: PTEN inhibitor bisperoxovanadium protects against noise-induced hearing loss

doi: 10.4103/1673-5374.358606

Figure Lengend Snippet: bpV prevents noise-induced reductions in PI3K-Akt signaling in OHCs. (A, B) Representative images showing the reduced levels of PI3K-p85α and -p110α (red, Alexa Fluor 594) in OHCs after noise exposure, as detected by immunofluorescence staining. Pretreatment with bpV protected against these effects. Scale bars: 20 μm. (C, D) Quantification of PI3K-p85α and -p110α levels in OHCs in noise (PTS) and noise + bpV (PTS + bpV) groups. (E, F) Representative images and quantification analyses showed that p-Akt levels were reduced in OHCs after noise exposure. (G) mRNA expression levels of PI3K and Akt were detected by real-time PCR in control, noise, and noise + bpV groups. (H) Protein expression of PI3K-p85α, -p110α, p-Akt and Akt were determined by western blotting. (I–K) Quantification of PI3K-p85α and -p110α relative to GAPDH and of p-Akt/Akt. Expression of p110α decreased after noise exposure compared with that in the control group. There was no change in total Akt level between the three groups. Data are presented as the mean ± SD. The experiment was repeated three times. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired Student’s t -test). bpV: Bisperoxovanadium; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IHC: inner hair cell; OHC: outer hair cell; p-Akt: phospho-Akt; PCR: polymerase chain reaction; PI3K: phosphoinositide-3 kinase; PTS: permanent threshold shift.

Article Snippet: The primary antibodies were monoclonal rabbit anti-PTEN (Cell Signaling Technology, Cat# 9188, RRID: AB_2253290), monoclonal rabbit anti-PI3K-p110α (Cell Signaling Technology, Cat# 4249, RRID: AB_2165248), polyclonal rabbit anti-PI3K-p85α (Cell Signaling Technology, Cat# 4292, RRID: AB_329869), monoclonal rabbit anti-phospho-Akt (Ser 473) (Cell Signaling Technology, Cat# 4060, RRID: AB_2315049) and polyclonal rabbit anti-4 hydroxynonenal (4HNE; Abcam, Cambridge, UK, Cat# ab46545, RRID: AB_722490).

Techniques: Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: PTEN inhibitor bisperoxovanadium protects against noise-induced hearing loss

doi: 10.4103/1673-5374.358606

Figure Lengend Snippet: Intratympanic delivery of PTEN siRNA attenuates noise-induced PTEN expression and hearing threshold changes and upregulates p85α level. (A, B) Immunolabeled PTEN (red, Alexa Fluor 594) in the cuticular plate and nucleus in basal OHCs after noise exposure in siControl (siCon) and siPTEN groups. (C, D) PTEN (red, Alexa Fluor 594) level was decreased in the nucleus and cuticular plate in the siPTEN-pretreated group after noise exposure. (E, F) The siPTEN group had upregulated p85α (red, Alexa Fluor 594) level in basal OHCs after noise exposure compared with the siCon group. Scale bars: 20 μm. (G, H) PTEN protein expression was detected by western blotting after noise exposure. (I) The siPTEN group had a reduced hearing threshold compared with that of the siCon group. Data are presented as the mean ± SD ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired Student’s t -test). ABR: Auditory brainstem response; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; OHC: outer hair cell; PTEN: Phosphatase and tensin homolog; siPTEN: PTEN silencing RNA.

Article Snippet: The primary antibodies were monoclonal rabbit anti-PTEN (Cell Signaling Technology, Cat# 9188, RRID: AB_2253290), monoclonal rabbit anti-PI3K-p110α (Cell Signaling Technology, Cat# 4249, RRID: AB_2165248), polyclonal rabbit anti-PI3K-p85α (Cell Signaling Technology, Cat# 4292, RRID: AB_329869), monoclonal rabbit anti-phospho-Akt (Ser 473) (Cell Signaling Technology, Cat# 4060, RRID: AB_2315049) and polyclonal rabbit anti-4 hydroxynonenal (4HNE; Abcam, Cambridge, UK, Cat# ab46545, RRID: AB_722490).

Techniques: Expressing, Immunolabeling, Western Blot

Journal: Mechanisms of ageing and development

Article Title: Cycloastragenol activation of telomerase improves β-Klotho protein level and attenuates age-related malfunctioning in ovarian tissues.

doi: 10.1016/j.mad.2022.111756

Figure Lengend Snippet: Fig. 5. Effect of cycloastragenol (CAG) and D-gal on cultured female embryo gonads and on adult mouse ovaries. (A) Representative images of hematoxylin and eosin-stained mouse ovaries obtained from mice belonging to the control, D-gal (200 mg/kg), and D-gal + CAG (20 mg/kg) groups. (B) Immunoblot analysis revealed that the protein levels of ER-α, Nr5a1, and Foxl2 in the D-gal-treated group were significantly downregulated when compared with those in the control and CAG- treated groups. β-actin was used as a loading control for western blotting analysis. Band intensities were quantified using ImageJ software and represented as histograms. The data are expressed as the mean ± SEM for the indicated proteins (n = 4 mice per group). (C) Western blotting analysis revealed that the protein levels of Klotho, Fgfr1, PI3K, and ERK1/2 in the D-gal-treated group were significantly (P < 0.05) downregulated when compared with those in the control and CAG- treated groups. (D) The gonads were obtained from 12.5-day embryos and cultured for 23 days. The data are expressed as the mean ± SEM for the indicated proteins in vitro (n = 6 gonads per group). (E) Immunofluorescence images of Klb (green fluorescent protein-labeled), and Fgfr1 (red fluorescent protein-labeled) in female embryonic gonads cultured for 22 days. The cells were counterstained with DAPI (blue) to visualize DNA. CAG treatment significantly improved Klb protein level, but fgfr1 remained non-significant. The data are indicated as the mean ± SEM for n = 4 mice per group. N.S, not significant; *P < 0.05; * *P < 0.01; * **P < 0.001.

Article Snippet: The following antibodies were used in this study: TERT (cat. # sc393013), β-Klotho (cat. # ARP53325_P050), FGFR1 (Thermo Scientific 13–3100), p-AKT (Cell Signaling, cat. # 9271), AKT (Cell Signaling technology cat. # 9272), p-mTOR (cat. # ab84400), RAGE (sc-80652), ER-α (Abcam, Cambridge, Cambs, UK cat. # ab3575), SF-1 (Abcam cat. # ab168380), FOXL2 (LifeSpan BioScience, Seattle, WA, USA cat. # LSB12865), p-ERK1/2 (Cell Signaling, cat. CST 9101 S), ERK1/2 (Cell Signaling, cat. CST 9102 S), PI3K (cat. # sc-374534), BCL-2 (cat. # sc783 Santa Cruz Biotechnology, Dallas, TX, USA), CASP3 (cat. # . sc1225, Santa Cruz Biotechnology, Dallas, TX, USA), NRF-1(sc-365651), KEAP-1(cat. # sc-515432), and β-actin (cat. # sc-47778 Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Cell Culture, Staining, Control, Western Blot, Software, In Vitro, Immunofluorescence, Labeling

Journal: Immunity, Inflammation and Disease

Article Title: Galectin‐9 alleviates acute graft‐versus‐host disease after haplo‐hematopoietic stem cell transplantation by regulating regulatory T cell/effector T cell imbalance

doi: 10.1002/iid3.1177

Figure Lengend Snippet: Gene expression profiles, functional analysis, and expression analysis of key proteins in PI3K/AKT/mTOR pathway in CD4 + T cell subsets. (A) Volcano plot of differentially expressed genes between patients with or without aGVHD. (B) GO analysis of differentially expressed genes. (C) KEGG analysis of differentially expressed genes. (D) Gene Set Enrichment Analysis (GSEA) was used to analyze the signaling pathways (PI3K/AKT/mTOR signaling pathway) enrichment in different groups. (E) PI3K/AKT/mTOR pathway protein expression in the peripheral blood was measured by western blot. Compared with the control group, * p < .05, ** p < .01; Compared with aGVHD(+) group, & p < .05, && p < .01. (F) Correlation analysis in patients without aGVHD. Data were analyzed with Spearman correlation analysis. (G) Galectin‐9 downregulates p‐PI3K in the Tim‐3 + CD4 + T cells in vitro. Tim‐3 + CD4 + T cells from patients with aGVHD were treated with or without rhGalectin‐9 for 48 h. The levels of p‐PI3K were determined by western blot. Compared with the PBS group, * p < .05, ** p < .01. Compared with the Galectin‐9 + IgG group, △ p < .05, △△ p < .01; Compared with Galectin‐9 + RAPA group, & p < .05, & & p < .01. (H) Analysis of IFN‐γ, IL‐4, IL‐17, and TGF‐β secretion by Tim‐3 + CD4 + T cells in vitro. Levels of IFN‐γ, IL‐4, IL‐17, and TGF‐β in culture supernatant were detected by ELISA. Compared with the PBS group, * p < .05, ** p < .01. Compared with the Galectin‐9 + IgG group, △ p < .05, △△ p < .01; Compared with the Galectin‐9 + RAPA group, & p < .05, & & p < .01. aGVHD, acute graft‐versus‐host disease; CBA, cytometric bead array; ELISA, enzyme‐linked immunosorbent assay; GO, Gene Ontology; IFN‐γ, interferon‐gamma; IL, interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; PBS, phosphate‐buffered saline; TGF‐transforming growth factor.

Article Snippet: ABclonal Technology provided the PI3K p85α (cat#: A11526), Phospho‐PI3K P85α (Y467/Y199/Y464) (cat#: AP0854), and Phospho‐mTOR‐S2448 (cat#: AP0115) primary antibodies.

Techniques: Expressing, Functional Assay, Western Blot, In Vitro, Enzyme-linked Immunosorbent Assay, Saline

Journal: Immunity, Inflammation and Disease

Article Title: Galectin‐9 alleviates acute graft‐versus‐host disease after haplo‐hematopoietic stem cell transplantation by regulating regulatory T cell/effector T cell imbalance

doi: 10.1002/iid3.1177

Figure Lengend Snippet: Schematic diagram illustrating the hypothesis of signaling regulation in aGVHD. In patients with aGVHD, the expression of Tim‐3 is significantly increased. Galectin‐9 binding to Tim‐3 may inhibit the activation of the PI3K/AKT pathway and enhance the function of Treg cells. On the other hand, TGF‐β promotes the differentiation of Treg cells through autocrine secretion, while TGF‐β induces the expression of Galectin‐9 in a paracrine manner. The increased Treg cells can inhibit the activation of Th1 and Th17 cells by secreting TGF‐β, thus alleviating aGVHD by inducing immune tolerance. aGVHD, acute graft‐versus‐host disease; IFN‐γ, interferon‐gamma; IL, interleukin; TGF‐transforming growth factor.

Article Snippet: ABclonal Technology provided the PI3K p85α (cat#: A11526), Phospho‐PI3K P85α (Y467/Y199/Y464) (cat#: AP0854), and Phospho‐mTOR‐S2448 (cat#: AP0115) primary antibodies.

Techniques: Expressing, Binding Assay, Activation Assay

Journal: FASEB BioAdvances

Article Title: NCAPD3 ‐mediated AKT activation regulates prostate cancer progression

doi: 10.1096/fba.2024-00073

Figure Lengend Snippet: NCAPD3 upregulated the phosphorylation level of AKT (T308) via JAK2/PI3K axis. (A) Protein levels of PI3K p110β, PI3K p85, AKT, PTEN, p‐PI3K p85, and p‐AKT (T308) assayed by Western blot in NCAPD3 overexpression or knockdown PCa cells. (B) Protein expressions of NCAPD3, AKT, p‐AKT (T308), FOXO1, and p‐FOXO1 (S256) in DU145 cells with transfection of NCAPD3 and treatment of LY294002 (20 μM). (C and D) Levels of JAK2 protein and mRNA expressions in NCAPD3‐overexpression DU145 cells and NCAPD3‐knockdown LNCaP cells. (E) Protein levels of NCAPD3, PI3K, p‐PI3K, AKT, and p‐AKT (T308) in DU145 cells with transfection of NCAPD3 and treatment of Z3 (30 μM). Values are means ± SE from n = 3 independent repetitions, * p < 0.05, ** p < 0.01, *** p < 0.001, based on Student's t ‐test.

Article Snippet: Antibodies against p‐FoxO1(Ser256) (Cat#AF3417), RICTOR (Cat#DF7530), RAPTOR (Cat#DF7527), PI3K p85α (Cat#AF6241), p‐PI3K p85α (Tyr607) (Cat#AF3241), and p‐STAT3(Y705) (Cat#AF3293) were from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Western Blot, Over Expression, Knockdown, Transfection

Journal: FASEB BioAdvances

Article Title: NCAPD3 ‐mediated AKT activation regulates prostate cancer progression

doi: 10.1096/fba.2024-00073

Figure Lengend Snippet: NCAPD3 increased the level of STAT3 and recruited more STAT3 to the promoters of EZH2 and JAK2 and then activated EZH2/NSD2/mTORC2 and JAK2/PI3K pathways, which phosphorylated AKT at Thr 308 and Ser 473. Moreover, there is a positive mutual activation between STAT3 and JAK2, further enhanced by NCAPD3 to promote PCa progression. The graph was drawn by Figdraw.

Article Snippet: Antibodies against p‐FoxO1(Ser256) (Cat#AF3417), RICTOR (Cat#DF7530), RAPTOR (Cat#DF7527), PI3K p85α (Cat#AF6241), p‐PI3K p85α (Tyr607) (Cat#AF3241), and p‐STAT3(Y705) (Cat#AF3293) were from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Activation Assay

Journal: Oncology reports

Article Title: YWHAZ promotes ovarian cancer metastasis by modulating glycolysis.

doi: 10.3892/or.2018.6920

Figure Lengend Snippet: Figure 5. Effect of YWHAZ on glycolysis metabolism in ovarian cancer cells. (A and B) The mRNA and protein expression of related glycolytic enzymes [lactate dehydrogenase A (LDHA), human platelet phosphofructokinase (PFKP) and glyceraldehyde‑3‑dehydrogenase (PGD)] was examined after (A) ES‑2 and (B) SKOV3 cells were transfected with siRNAs targeting YWHAZ by real‑time PCR and western blot analysis. (C and D) The expression of glycolytic metabolites [lactate production, extracellular acidification rate (ECAR) and nicotinamide adenine dinucleotide (NADH) production] after (C) ES‑2 and (D) SKOV3 cells were transfected with siRNAs targeting YWHAZ. (E and F) The protein expression of PI3K, pAkt1 and vimentin were detected after ES‑2 and SKOV3 cells were transfected with (E) siRNAs targeting YWHAZ or (F) lenti‑YWHAZ overexpressing plasmid. Tubulin was used as an internal control. Values are means mean ± SD. **P<0.01.

Article Snippet: Antibodies used in this study included YWHAZ (cat. no. ab51129; Abcam, Cambridge, MA, USA), AKT (cat. no. ab18785; Abcam), p-AKT1 (cat. no. ab133458; Abcam), PI3K-p85α (cat. no. 13666), vimentin (cat. no. 5741), LDHA (cat. no. 2012), PFKP (cat. no. 5412), tubulin (cat. no. 2146; all were from Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

doi: 10.1038/ncomms8721

Figure Lengend Snippet: ( a ) Acute EGF treatment promoted PTEN, but diminished TNS3 binding to DLC1. MCF10A cells were serum starved and incubated with EGF or not for 5, 15 or 30 min before IP and IB with the specified antibodies. WCL, whole-cell lysate. ( b ) Acute EGF treatment gradually promoted PI3K binding to TNS3 at the expense of PTEN. MCF10A cells under the same conditions as in a were subjected to IP/IB using the indicated antibodies. An anti-p85 antibody was used to detect PI3K. ( c ) The domain structures of PTEN and TNS3–ABD. The two Thr phosphorylation sites in the PTEN-C2 domain and the equivalent residues in the ABD C2 domain are identified. ( d ) EGF treatment of HEK293 cells enhanced PTEN, but abolished TNS3–ABD binding to DLC1. PTEN and TNS3–ABD were expressed as GFP fusions and DLC1 with a FLAG-tag to facilitate IP/IB analysis. ( e ) A PTEN–PI3K(p85) complex was replaced by a TNS3(ABD)–PI3K(p85) complex in response to EGF treatment in HEK293 cells overexpressing these proteins. ( f ) The C2 domain, but not the amino-terminal region of TNS3–ABD, mediated binding to DLC1. HEK293 cells co-expressing FLAG-DLC1 and GFP fusion TNS3–ABD, ABD-N or the -C2 domain were subjected to IP/IB analysis using anti-GFP and anti-FLAG antibodies. ( g ) The C2 domain was responsible for TNS3–ABD binding to PI3K–p85. GFP-ABD, -ABD-N or -C2 was examined for binding to endogenous p85 in HEK293 cells. ( h ) The C2, but not the catalytic domain (CAT), mediated binding of PTEN to DLC1. Full-length PTEN, or its catalytic or C2 domain was expressed as GFP fusion and examined for binding to FLAG-DLC1 in HEK293 cells. ( i ) The PTEN-C2, but not the catalytic domain, bound to endogenous p85 in HEK293 cells. Cells used in f – i were cultured in serum-containing media. Data shown are representative of three independent experiments.

Article Snippet: Rabbit anti-p44/42 MAPK (Erk1/2; cat # 9102), mouse anti-Phospho-p44/42 MAPK (Thr202/Tyr204; cat # 9106), mouse anti-Phospho-Tyrosine(cat # 9411S) and mouse anti-Phospho-Threonine (cat # 9386)antibodies were purchased from Cell Signaling Technology Inc. Rabbit anti-PI3K p110α (cat # 04-399), mouse anti-PI3K p85α (cat # 05-212) and mouse anti-Phosphoserine (cat # 05-1000) antibodies were obtained from Millipore.

Techniques: Binding Assay, Incubation, FLAG-tag, Expressing, Cell Culture

Journal: Nature Communications

Article Title: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

doi: 10.1038/ncomms8721

Figure Lengend Snippet: ( a ) Thr319 in PTEN, and the equivalent Tyr321 in TNS3–ABD, controlled their dynamic interactions with DLC1 in response to EGF treatment. HEK293 cells co-expressing DLC1 and wt PTEN or a mutant (T319A, T319E or T319Y), or wt TNS3–ABD or the Y321T mutant were subjected to IP/IB to examine their interactions in the absence (−) or presence (+) of EGF stimulation. DLC1 was tagged with FLAG; PTEN, TNS3 or the mutants were fused to GFP. ( b ) The same panel of proteins as in a were examined for binding to endogenous PI3K (via p85). ( c , d ) Thr321 in PTEN and Thr323 in TNS3–ABD play an important role in dictating binding to DLC1 ( c ) or PI3K–p85 ( d ). ( e , f ) Recombinant GST–PTEN, GST–TNS3–ABD or an indicated mutant was used to pull down FLAG-DLC1 ( e ) or FLAG-p85 ( f ) from HEK293 cells. The bound proteins were detected by anti-FLAG IBs. ( g ) A summary of binding specificities displayed by the panel of mutants used in a – f and in . Data shown are representative of three independent experiments.

Article Snippet: Rabbit anti-p44/42 MAPK (Erk1/2; cat # 9102), mouse anti-Phospho-p44/42 MAPK (Thr202/Tyr204; cat # 9106), mouse anti-Phospho-Tyrosine(cat # 9411S) and mouse anti-Phospho-Threonine (cat # 9386)antibodies were purchased from Cell Signaling Technology Inc. Rabbit anti-PI3K p110α (cat # 04-399), mouse anti-PI3K p85α (cat # 05-212) and mouse anti-Phosphoserine (cat # 05-1000) antibodies were obtained from Millipore.

Techniques: Expressing, Mutagenesis, Binding Assay, Recombinant

Journal: Nature Communications

Article Title: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

doi: 10.1038/ncomms8721

Figure Lengend Snippet: ( a ) Depletion of TNS3 from MCF10A activated RhoA, but not Rac1. GTP-bound RhoA or Rac1 was pulled down (PD) with Rhotekin-RBD or PAK-PBD agarose beads from cell lysates and blotted with an anti-RhoA or anti-Rac1 antibody. WCL IB was included to show the total levels of TNS3, RhoA and Rac1. ( b ) Depletion of PTEN augmented the activity of Rac1, but not RhoA. The same set of experiments as in a were carried out in MCF10A cells transfected with a PTEN-specific siRNA or a scrambled oligo (siCtrl). ( c ) Overexpression of TNS3–ABD, but not PTEN nor a mutant of either protein, led to inactivation of RhoA. HEK293 cells expressing GFP fusion TNS3–ABD, PTEN or the indicated mutants were subjected to a Rhotekin-PD assay. ( d ) PTEN and its point mutants, but neither a PTEN truncation mutant missing the catalytic domain (▵CAT) nor TNS3–ABD and its point mutants, repressed Rac1 activity. A PAK-PBD PD assay was used to assess Rac1 activity in HEK293 cells co-expressing PI3K (that is, FLAG-p85 plus Myc-p110) and the indicated PTEN or TNS3–ABD proteins. ( e ) The specificity-switching mutant, TNS3–ABD–Y321T, but neither PTEN, PTEN–T319Y, nor TNS3–ABD, inactivated RhoA. DLC1-expressing HEK293 cells were serum starved and treated with EGF for 30 min before the Rhotekin-PD assay. ( f ) wt PTEN and the specificity-switching mutant, PTEN–T319Y, but neither TNS3–ABD nor the ABD–Y321T mutant, inhibited the gross activation of Rac1. Cells were treated the same way as in e . Data shown are representative of three independent experiments.

Article Snippet: Rabbit anti-p44/42 MAPK (Erk1/2; cat # 9102), mouse anti-Phospho-p44/42 MAPK (Thr202/Tyr204; cat # 9106), mouse anti-Phospho-Tyrosine(cat # 9411S) and mouse anti-Phospho-Threonine (cat # 9386)antibodies were purchased from Cell Signaling Technology Inc. Rabbit anti-PI3K p110α (cat # 04-399), mouse anti-PI3K p85α (cat # 05-212) and mouse anti-Phosphoserine (cat # 05-1000) antibodies were obtained from Millipore.

Techniques: Activity Assay, Transfection, Over Expression, Mutagenesis, Expressing, Activation Assay

Journal: Nature Communications

Article Title: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

doi: 10.1038/ncomms8721

Figure Lengend Snippet: ( a ) Front–rear-polarized localization of PI3K and PTEN. EGF-treated MCF10A cells were co-immunostained for PTEN (blue) and PI3K-p110 (green). Actin was stained with rhodamine-phallodin (red). ( b ) Front–rear-polarized localization of TNS3 and DLC1 in cells under the same condition as in a . ( c ) TNS3 and PI3K-p110 co-localized to the leading edge of migrating cells. Cells as in a were immunostained for TNS3 (blue) and PI3K-p110 (green). ( d ) Rac1-GTP and TNS3 co-localized to the lamellipodial protrusion at the leading edge of a migrating cell. Cells were co-stained for Rac1-GTP (blue), TNS3 (green) and actin (red). ( e ) DLC1 (blue) and PTEN (green) co-localized to the posterior of a migrating cell. ( f ) RhoA-GTP (blue) and PTEN (green) co-localized to the posterior of a migrating cell. Images shown are representative of three independent experiments. Arrow indicates direction of migration. Scale bar, 10 μm.

Article Snippet: Rabbit anti-p44/42 MAPK (Erk1/2; cat # 9102), mouse anti-Phospho-p44/42 MAPK (Thr202/Tyr204; cat # 9106), mouse anti-Phospho-Tyrosine(cat # 9411S) and mouse anti-Phospho-Threonine (cat # 9386)antibodies were purchased from Cell Signaling Technology Inc. Rabbit anti-PI3K p110α (cat # 04-399), mouse anti-PI3K p85α (cat # 05-212) and mouse anti-Phosphoserine (cat # 05-1000) antibodies were obtained from Millipore.

Techniques: Staining, Migration

Journal: Nature Communications

Article Title: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

doi: 10.1038/ncomms8721

Figure Lengend Snippet: ( a ) Depletion of TNS3 markedly enhanced actin stress fibre formation, whereas the knockdown of PTEN dramatically promoted lamellipodial formation. MCF10A cells were transfected with siRNA specific for TNS3 or PTEN and serum starved before immunostaining and confocal imaging: TNS3 and PTEN, green; actin, red. Each microscopic field contains one non-transfected cell identified by positive IF for TNS3 or PTEN (marked by asterisks). ( b – e ) Abnormal cell morphology engendered by the expression of a PTEN, TNS3, Mek1/2 or Erk1/2mutant. MCF10A cells were transfected with plasmids encoding the indicated proteins, serum starved and imaged after 30 min incubation with EGF (+EGF) or buffer (−EGF). Note that the green fluorescence in b – e indicates transfected cells. ( f , g ) Phase-contrast images of HeLa or HeLa DLC1 cells at 0 and 16 h after wounding by scratch. Right, confocal images of the same cells with actin staining (red). Cells were cultured in serum-free medium with EGF. Images shown in a – g are representative of four independent experiments. Scale bar, 10 μm (white), 100 μm (red). ( h , i ) The defective P-switch in HeLa was restored in the HeLa DLC1 cells that stably express DLC1. Both the HeLa and HeLa DLC1 cells, treated (+) or not (−) with EGF, were subjected to IP/IB to evaluate the dynamic binding among TNS3, PTEN, PI3K and DLC1.

Article Snippet: Rabbit anti-p44/42 MAPK (Erk1/2; cat # 9102), mouse anti-Phospho-p44/42 MAPK (Thr202/Tyr204; cat # 9106), mouse anti-Phospho-Tyrosine(cat # 9411S) and mouse anti-Phospho-Threonine (cat # 9386)antibodies were purchased from Cell Signaling Technology Inc. Rabbit anti-PI3K p110α (cat # 04-399), mouse anti-PI3K p85α (cat # 05-212) and mouse anti-Phosphoserine (cat # 05-1000) antibodies were obtained from Millipore.

Techniques: Transfection, Immunostaining, Imaging, Expressing, Incubation, Fluorescence, Staining, Cell Culture, Stable Transfection, Binding Assay

Journal: Nature Communications

Article Title: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

doi: 10.1038/ncomms8721

Figure Lengend Snippet: ( a ) A western blot showing the expression of PDGFR-β in MDA-MB-231, but not in MCF10A. ( b ) Acute PDGF treatment (30 min) following serum starvation led to phosphorylation of Erk1/2 and activation of RhoA and Rac1. ( c ) PDGF regulates the dynamic interactions of DLC1, TNS3, PTEN and PI3K. ( d ) A western blot showing the knockdown of DLC1 in MDA-MB-231 cells. ( e ) A ‘defective' P-switch in DLC1-depleted MDA-MB-231: PI3K–p85 was found to bind both PTEN and TNS3 in the absence of DLC1 following PDGF stimulation. ( f ) Phase-contrast images of MCF10A and MDA-MB-231 cells transfected with control or DLC1-specific siRNA at 0 and 16 h after wounding by scratch. Cells were cultured in serum-free medium with PDGF. ( g ) Confocal images of MDA-MB-231 cells with or without depletion of DLC1 showing the distinct patterns of actin network at the edge of the scratch wound. Scale bar, 10 μm (white), 100 μm (red). Data shown are representative of three independent experiments.

Article Snippet: Rabbit anti-p44/42 MAPK (Erk1/2; cat # 9102), mouse anti-Phospho-p44/42 MAPK (Thr202/Tyr204; cat # 9106), mouse anti-Phospho-Tyrosine(cat # 9411S) and mouse anti-Phospho-Threonine (cat # 9386)antibodies were purchased from Cell Signaling Technology Inc. Rabbit anti-PI3K p110α (cat # 04-399), mouse anti-PI3K p85α (cat # 05-212) and mouse anti-Phosphoserine (cat # 05-1000) antibodies were obtained from Millipore.

Techniques: Western Blot, Expressing, Activation Assay, Transfection, Cell Culture

Journal: Nature Communications

Article Title: A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration

doi: 10.1038/ncomms8721

Figure Lengend Snippet: The model depicts how a molecular switch comprising TNS3, DLC1, PI3K and PTEN controls the directional migration of epithelial cells via spatiotemporal activation of Rac1 and RhoA. a , without EGF stimulation; b , with EGF.

Article Snippet: Rabbit anti-p44/42 MAPK (Erk1/2; cat # 9102), mouse anti-Phospho-p44/42 MAPK (Thr202/Tyr204; cat # 9106), mouse anti-Phospho-Tyrosine(cat # 9411S) and mouse anti-Phospho-Threonine (cat # 9386)antibodies were purchased from Cell Signaling Technology Inc. Rabbit anti-PI3K p110α (cat # 04-399), mouse anti-PI3K p85α (cat # 05-212) and mouse anti-Phosphoserine (cat # 05-1000) antibodies were obtained from Millipore.

Techniques: Migration, Activation Assay