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  • 99
    Millipore ly294002
    Deletion of IFT80 inhibited DPSC proliferation via down-regulation of FGF2–FGFR1–PI3K–AKT signaling. a Western blot analysis of AKT phosphorylation with FGF2 (10 ng/mL) stimulation in IFT80 f/f and IFT80 d/d DPSCs. AKT phosphorylation level is normalized to AKT ( n = 3). PD PD173074 (1 μM); API API-2 (1 μM, AKT inhibitor). b Quantification of protein expression from a . AKT phosphorylation level was normalized AKT ( n = 3). c Proliferation (MTS) assay of IFT80 f/f and IFT80 d/d DPSCs treated with FGF2 together with PD173074 (PD, 1 μM), <t>LY294002</t> (LY, 15 μM, PI3K inhibitor), API-2 (API, 1 μM), or rapamycin (RAP, 0.5 μM, mTOR inhibitor). The starting cell density is 100 cells per mm 2 ( n = 3, triplicates per group). Data are expressed as mean ± SEM; ns, not statistically significant; *** p
    Ly294002, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9740 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc ly294002
    EGF-like repeats of Npnt regulate EGF signaling pathway by regulating phosphorylation of Akt. ( A ) Western blotting of P-Akt, Akt, <t>PI3K,</t> P-PLCγ, P-Stat5, P-Erk1/2, Erk1/2, and Gapdh in M3H1 cells transfected with Npnt-FL or Npnt-ΔEGF, and cultured for 48 hours. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1B . ( B ) Western blotting of P-Akt and Akt in M3H1 cells with or without recombinant Npnt treatment for 60 minutes. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1C . ( C ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL and treated with different doses of PI3K inhibitor (LY294002). The ratio was calculated as Sox2 positive cells/DAPI-stained nuclei. ( D ) BrdU incorporation in M3H1 cells treated with different doses of LY294002. The ratio was calculated as BrdU-positive cells/DAPI-stained nuclei. *P
    Ly294002, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3884 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals ly294002
    EGF-like repeats of Npnt regulate EGF signaling pathway by regulating phosphorylation of Akt. ( A ) Western blotting of P-Akt, Akt, <t>PI3K,</t> P-PLCγ, P-Stat5, P-Erk1/2, Erk1/2, and Gapdh in M3H1 cells transfected with Npnt-FL or Npnt-ΔEGF, and cultured for 48 hours. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1B . ( B ) Western blotting of P-Akt and Akt in M3H1 cells with or without recombinant Npnt treatment for 60 minutes. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1C . ( C ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL and treated with different doses of PI3K inhibitor (LY294002). The ratio was calculated as Sox2 positive cells/DAPI-stained nuclei. ( D ) BrdU incorporation in M3H1 cells treated with different doses of LY294002. The ratio was calculated as BrdU-positive cells/DAPI-stained nuclei. *P
    Ly294002, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 874 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    cayman chemical ly294002
    Scanning electron microscopy analysis. Representative images of NT2D1 cells cultured for 24 h in control conditions, or treated with HGF, <t>LY294002,</t> or their combination. Scale bar: 10 μm.
    Ly294002, supplied by cayman chemical, used in various techniques. Bioz Stars score: 92/100, based on 780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pi3k inhibitor ly294002
    Scanning electron microscopy analysis. Representative images of NT2D1 cells cultured for 24 h in control conditions, or treated with HGF, <t>LY294002,</t> or their combination. Scale bar: 10 μm.
    Pi3k Inhibitor Ly294002, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Beyotime ly294002
    α7nAChR expressed in THP-1 derived macrophages (TMs) inhibits the migration of CRC cells through the JAK2/STAT3 signaling pathway. TMs were treated with or without α-bungarotoxin (α-Btx) for 6 h and then with DMSO (control), AG490, <t>LY294002</t> and Bay 11–7082 for 1 h, respectively. Subsequently, LoVo cells were co-cultured with TM in 24-well Transwell plates with 8 µm polycarbonate membrane filters for 36 h for the migration assay. (A) Upper panel: Representative images show the migration of LoVo cells under the condition that the TMs did not have the α-Btx treatment and were treated with AG490, LY294002 and Bay 11–7082 only, respectively. Lower panel: Representative images show the migration of LoVo cells under the condition that the TMs were treated with α-Btx only, or dually treated with AG490, LY294002 and Bay 11–7082, respectively. (B) Number of migrated LoVo cells are presented as the mean ± SD of six images (n=6) of three separate experiments. Significance of differences between groups was determined by Student's t-test. P-value
    Ly294002, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 448 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Tocris ly294002
    α7nAChR expressed in THP-1 derived macrophages (TMs) inhibits the migration of CRC cells through the JAK2/STAT3 signaling pathway. TMs were treated with or without α-bungarotoxin (α-Btx) for 6 h and then with DMSO (control), AG490, <t>LY294002</t> and Bay 11–7082 for 1 h, respectively. Subsequently, LoVo cells were co-cultured with TM in 24-well Transwell plates with 8 µm polycarbonate membrane filters for 36 h for the migration assay. (A) Upper panel: Representative images show the migration of LoVo cells under the condition that the TMs did not have the α-Btx treatment and were treated with AG490, LY294002 and Bay 11–7082 only, respectively. Lower panel: Representative images show the migration of LoVo cells under the condition that the TMs were treated with α-Btx only, or dually treated with AG490, LY294002 and Bay 11–7082, respectively. (B) Number of migrated LoVo cells are presented as the mean ± SD of six images (n=6) of three separate experiments. Significance of differences between groups was determined by Student's t-test. P-value
    Ly294002, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH ly294002
    The effect of STS on phosphorylated FOXO1, FOXO3A, and FOXO4 levels. Left: a representative Western-blot from left ventricle tissues isolated from each group showing the effect of STS on the levels of phosphorylated (p)-FOXO1, p-FOXO3A, p-FOXO4, total t-FOXO1, t-FOXO3A, and t-FOXO4. Right: the quantitative estimation of p-FOXO1, p-FOXO3A, and p-FOXO4 levels are shown as the relative OD values in STS and <t>STS+LY294002</t> groups compared to the NS group. b P
    Ly294002, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 522 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories ly294002
    RT-PCR analysis of GS-2, GBM8, and GBM6 cell lines treated with temozolomide, <t>LY294002,</t> and everolimus. CA-IX mRNA expression was significantly decreased following LY294002 treatment in each cell line: 10% ± 2% of control in GS-2 ( P = 2 ×
    Ly294002, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher ly294002
    Modulation of PI3K-Akt pathway affects autophagic and phagocytic activity as well as inflammatory response in microglial cells. a A representative Western blot image of lysates of BV2 cells treated with <t>LY294002</t> (LY) (0.1 µM – 10µM), and b with LY294002 together with LPS. c Quantification revealed that LY294002 decreased Akt S473 phosphorylation levels dose-dependently in cells treated with LY294002 and together with LPS and LY294002. d Quantification of LC3B II and I levels revealed a significant increase in LCB3II/I ratio by LY294002. e Phagocytic activity in BV2 cells was assessed using pHrodo bioparticles and fluorescence emission was measured in the IncuCyte live cell imaging device in every 15 min. BV2 cells were treated with four different concentrations of PI3K inhibitor LY294002 (5µM – 50µM) and with 5µM Cytochalasin D (CytD). LY294002 decreased phagocytic activity dose-dependently (p
    Ly294002, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA ly294002
    PI3K signaling regulates B cell commitment and plasticity. ( a ) Cells from bm-derived wt and p110dKO pre-B cell lines were cultured either in the presence of IL-7, M-CSF, or without cytokines and analyzed by flow cytometry. Numbers indicate percentages of cells in the respective regions at day 7 after beginning of treatment. (Data are representative of at least 3 independent experiments). ( b ) FACS-analysis of CD11b expression of measured in cells from bm-derived wt and p110dKO pre-B cell lines cultured in the presence of IL-7 (upper panel). p110dKO cells were cultured in IL-7 or M-CSF supplemented medium and CD11b expression was assessed by FACS analysis at day 8 after beginning of treatment (lower panel). ( c ) Cells from a bm-derived SLP-65-deficient pre-B cell line were treated with <t>LY294002</t> (30 μM) or solvent (DMSO) for 16 h and subsequently cultured in medium supplemented with M-CSF. Csf1r and Notch1 mRNA levels were determined with specific primers by qRT-PCR using the SYBR-Green detection method (left panel). Results are shown as mean ± SD of 2 independent analyses, run as duplicates. Statistical significance was calculated using the Mann-Whitney Test or the t-Test. Cells from the respective culture conditions were further analyzed by flow cytometry (right panel).
    Ly294002, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pi3k inhibitor
    Inhibition of <t>PI3K</t> disrupts Akt/HIF-1 signaling and recreates liver IRI in Keap1HKO OLTs. Groups of WT or Keap1HKO liver donor mice were pre-treated with Ly294002 or DMSO (-1 h). (A) sALT levels (IU/L): (□) sham; ( ) WT+DMSO; ( ) WT+ Lly294002;
    Pi3k Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co ly294002
    NOX4 promotes NSCLC progression through activating PI3K/Akt pathway in vitro (A-C) NOX4 overexpressing A549 and H460 tumors-bearing animals were treated with <t>LY294002</t> (25 mg/kg, every four days, i.p.).The growth curves of tumor (A), number of metastatic nodules on the surface of the lungs of mice (B), and Kaplan-Meier curves for illustration of the survival periods (C) were respectively shown. Bars are mean ± SD. *Significantly different from vector control, P
    Ly294002, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam ly294002
    Phospho-Akt and Akt expression, Phospho-GSK-3 β and GSK-3 β expression and Phospho-ERK1/2 and ERK1/2 expression in MIRI rats. ( A ) Phospho-Akt and Akt assay (1–4, represent results of Sham, MIRI, H-TFDM + MIRI + <t>LY294002,</t> H-TFDM + MIRI group, respectively, ## P
    Ly294002, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ApexBio ly294002
    Phospho-Akt and Akt expression, Phospho-GSK-3 β and GSK-3 β expression and Phospho-ERK1/2 and ERK1/2 expression in MIRI rats. ( A ) Phospho-Akt and Akt assay (1–4, represent results of Sham, MIRI, H-TFDM + MIRI + <t>LY294002,</t> H-TFDM + MIRI group, respectively, ## P
    Ly294002, supplied by ApexBio, used in various techniques. Bioz Stars score: 90/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    InvivoGen ly294002
    BCP crystals induce expression of S100A8, S100A12, and MMP1 in primary macrophages in a Syk-, PI3K-, and ERK-dependent manner. Human macrophages were pre-treated with a piceatannol (50 μM, 100 μM), b <t>LY294002</t> (25 μM, 50 μM), or c PD98059 (5 μM, 10 μM) for 30 mins prior to stimulation with BCP crystals for 24 h. mRNA levels of S100A8, S100A12, MMP1, and TIMP1 were analysed by real-time PCR. Results indicate mean (± SEM) of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
    Ly294002, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    LY294002 is a selective phosphatidylinositol 3 kinase PI3K inhibitor with a 2 7 fold greater potency than quercetin LY294002 inhibits purified PI3K with an IC of 1 4 µM
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    N/A
    LY303511 an inactive analogue of LY294002 is a mTOR inhibitor that did not inhibit PI3 K
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    N/A
    LY294002 is a morpholine containing potent reversible inhibitor of phosphoinositide 3 kinases PI3Ks and BETproteins It is reported to be promote the differentiation of insulin producing cells from embryonic cells
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    Image Search Results


    Deletion of IFT80 inhibited DPSC proliferation via down-regulation of FGF2–FGFR1–PI3K–AKT signaling. a Western blot analysis of AKT phosphorylation with FGF2 (10 ng/mL) stimulation in IFT80 f/f and IFT80 d/d DPSCs. AKT phosphorylation level is normalized to AKT ( n = 3). PD PD173074 (1 μM); API API-2 (1 μM, AKT inhibitor). b Quantification of protein expression from a . AKT phosphorylation level was normalized AKT ( n = 3). c Proliferation (MTS) assay of IFT80 f/f and IFT80 d/d DPSCs treated with FGF2 together with PD173074 (PD, 1 μM), LY294002 (LY, 15 μM, PI3K inhibitor), API-2 (API, 1 μM), or rapamycin (RAP, 0.5 μM, mTOR inhibitor). The starting cell density is 100 cells per mm 2 ( n = 3, triplicates per group). Data are expressed as mean ± SEM; ns, not statistically significant; *** p

    Journal: Cell Death & Disease

    Article Title: IFT80 is required for stem cell proliferation, differentiation, and odontoblast polarization during tooth development

    doi: 10.1038/s41419-018-0951-9

    Figure Lengend Snippet: Deletion of IFT80 inhibited DPSC proliferation via down-regulation of FGF2–FGFR1–PI3K–AKT signaling. a Western blot analysis of AKT phosphorylation with FGF2 (10 ng/mL) stimulation in IFT80 f/f and IFT80 d/d DPSCs. AKT phosphorylation level is normalized to AKT ( n = 3). PD PD173074 (1 μM); API API-2 (1 μM, AKT inhibitor). b Quantification of protein expression from a . AKT phosphorylation level was normalized AKT ( n = 3). c Proliferation (MTS) assay of IFT80 f/f and IFT80 d/d DPSCs treated with FGF2 together with PD173074 (PD, 1 μM), LY294002 (LY, 15 μM, PI3K inhibitor), API-2 (API, 1 μM), or rapamycin (RAP, 0.5 μM, mTOR inhibitor). The starting cell density is 100 cells per mm 2 ( n = 3, triplicates per group). Data are expressed as mean ± SEM; ns, not statistically significant; *** p

    Article Snippet: LY294002 (15 μM, Sigma, L9908), API-2 (1 μM, Tocris, 2151), rapamycin (0.5 μM, Selleckchem, S1039) were employed to inhibit PI3K, AKT, and mTOR, respectively.

    Techniques: Western Blot, Expressing, MTS Assay

    EGF-like repeats of Npnt regulate EGF signaling pathway by regulating phosphorylation of Akt. ( A ) Western blotting of P-Akt, Akt, PI3K, P-PLCγ, P-Stat5, P-Erk1/2, Erk1/2, and Gapdh in M3H1 cells transfected with Npnt-FL or Npnt-ΔEGF, and cultured for 48 hours. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1B . ( B ) Western blotting of P-Akt and Akt in M3H1 cells with or without recombinant Npnt treatment for 60 minutes. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1C . ( C ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL and treated with different doses of PI3K inhibitor (LY294002). The ratio was calculated as Sox2 positive cells/DAPI-stained nuclei. ( D ) BrdU incorporation in M3H1 cells treated with different doses of LY294002. The ratio was calculated as BrdU-positive cells/DAPI-stained nuclei. *P

    Journal: Scientific Reports

    Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

    doi: 10.1038/srep45181

    Figure Lengend Snippet: EGF-like repeats of Npnt regulate EGF signaling pathway by regulating phosphorylation of Akt. ( A ) Western blotting of P-Akt, Akt, PI3K, P-PLCγ, P-Stat5, P-Erk1/2, Erk1/2, and Gapdh in M3H1 cells transfected with Npnt-FL or Npnt-ΔEGF, and cultured for 48 hours. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1B . ( B ) Western blotting of P-Akt and Akt in M3H1 cells with or without recombinant Npnt treatment for 60 minutes. Gapdh was used as the internal control. The full-length blots are presented in Supplementary Figure 1C . ( C ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL and treated with different doses of PI3K inhibitor (LY294002). The ratio was calculated as Sox2 positive cells/DAPI-stained nuclei. ( D ) BrdU incorporation in M3H1 cells treated with different doses of LY294002. The ratio was calculated as BrdU-positive cells/DAPI-stained nuclei. *P

    Article Snippet: For a PI3K inhibiting assay, a PI3K inhibitor (LY294002, Cell Signaling Technology) was used at concentrations ranging from 0-50 μM.

    Techniques: Western Blot, Transfection, Cell Culture, Recombinant, Staining, BrdU Incorporation Assay

    Scanning electron microscopy analysis. Representative images of NT2D1 cells cultured for 24 h in control conditions, or treated with HGF, LY294002, or their combination. Scale bar: 10 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

    doi: 10.3390/ijms21228669

    Figure Lengend Snippet: Scanning electron microscopy analysis. Representative images of NT2D1 cells cultured for 24 h in control conditions, or treated with HGF, LY294002, or their combination. Scale bar: 10 μm.

    Article Snippet: Cell Death AnalysisCells, cultured as above, were treated with LY294002 at different concentrations (1, 5, 10, 15 µM) for 48 h. Cell death was evaluated by flow cytometry using propidium iodide (PI) exclusion assay: 2 µg/mL of PI solution (Sigma-Aldrich, cat. P4864, St. Louis, MO, USA) was added to each sample and PI fluorescence was determined by FACS (CyAn ADP, Beckman Coulter, Fullerton, CA, USA).

    Techniques: Electron Microscopy, Cell Culture

    ( I ) Cell death Flow Cytometry nalysis. Graphical representation of the percentage of live cells obtained by culturing NT2D1 cells with different concentrations of LY294002 for 48 h (* p

    Journal: International Journal of Molecular Sciences

    Article Title: The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

    doi: 10.3390/ijms21228669

    Figure Lengend Snippet: ( I ) Cell death Flow Cytometry nalysis. Graphical representation of the percentage of live cells obtained by culturing NT2D1 cells with different concentrations of LY294002 for 48 h (* p

    Article Snippet: Cell Death AnalysisCells, cultured as above, were treated with LY294002 at different concentrations (1, 5, 10, 15 µM) for 48 h. Cell death was evaluated by flow cytometry using propidium iodide (PI) exclusion assay: 2 µg/mL of PI solution (Sigma-Aldrich, cat. P4864, St. Louis, MO, USA) was added to each sample and PI fluorescence was determined by FACS (CyAn ADP, Beckman Coulter, Fullerton, CA, USA).

    Techniques: Flow Cytometry

    Effect of LY294002 on cell NT2D1 cell migration. ( I ) Quantitative analysis of chemoattracted NT2D1 cells. The values were calculated as “fold change” (±S.E.M.) compared to the control, which was considered as 1. The use of LY294002 in combination with HGF abrogates the migratory effect induced by HGF. ( II ) Representative images of NT2D1 cell migration. Images were recorded at 40× magnification. ( III ) Table illustrating the number of migrating cells/filter (* p

    Journal: International Journal of Molecular Sciences

    Article Title: The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

    doi: 10.3390/ijms21228669

    Figure Lengend Snippet: Effect of LY294002 on cell NT2D1 cell migration. ( I ) Quantitative analysis of chemoattracted NT2D1 cells. The values were calculated as “fold change” (±S.E.M.) compared to the control, which was considered as 1. The use of LY294002 in combination with HGF abrogates the migratory effect induced by HGF. ( II ) Representative images of NT2D1 cell migration. Images were recorded at 40× magnification. ( III ) Table illustrating the number of migrating cells/filter (* p

    Article Snippet: Cell Death AnalysisCells, cultured as above, were treated with LY294002 at different concentrations (1, 5, 10, 15 µM) for 48 h. Cell death was evaluated by flow cytometry using propidium iodide (PI) exclusion assay: 2 µg/mL of PI solution (Sigma-Aldrich, cat. P4864, St. Louis, MO, USA) was added to each sample and PI fluorescence was determined by FACS (CyAn ADP, Beckman Coulter, Fullerton, CA, USA).

    Techniques: Migration

    Effect of LY294002 on NT2D1 cell collective migration. ( I ) Quantitative analysis of wound closure after 24 h ( a ) and 48 h ( b ). Data are expressed as the mean percentage of residual open area compared with the respective T0 condition. At 24 h, the decrease of open area in HGF-treated cells was not statistically significant compared with the control condition, but the closure was almost complete at 48 h (a vs. b, p

    Journal: International Journal of Molecular Sciences

    Article Title: The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

    doi: 10.3390/ijms21228669

    Figure Lengend Snippet: Effect of LY294002 on NT2D1 cell collective migration. ( I ) Quantitative analysis of wound closure after 24 h ( a ) and 48 h ( b ). Data are expressed as the mean percentage of residual open area compared with the respective T0 condition. At 24 h, the decrease of open area in HGF-treated cells was not statistically significant compared with the control condition, but the closure was almost complete at 48 h (a vs. b, p

    Article Snippet: Cell Death AnalysisCells, cultured as above, were treated with LY294002 at different concentrations (1, 5, 10, 15 µM) for 48 h. Cell death was evaluated by flow cytometry using propidium iodide (PI) exclusion assay: 2 µg/mL of PI solution (Sigma-Aldrich, cat. P4864, St. Louis, MO, USA) was added to each sample and PI fluorescence was determined by FACS (CyAn ADP, Beckman Coulter, Fullerton, CA, USA).

    Techniques: Migration

    Effect of LY294002 on NT2D1 cell invasion. ( I ) Quantitative analysis of invading cells. Results are expressed as fold change (±S.E.M.) and the control condition is considered as 1 (b vs. a; p

    Journal: International Journal of Molecular Sciences

    Article Title: The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

    doi: 10.3390/ijms21228669

    Figure Lengend Snippet: Effect of LY294002 on NT2D1 cell invasion. ( I ) Quantitative analysis of invading cells. Results are expressed as fold change (±S.E.M.) and the control condition is considered as 1 (b vs. a; p

    Article Snippet: Cell Death AnalysisCells, cultured as above, were treated with LY294002 at different concentrations (1, 5, 10, 15 µM) for 48 h. Cell death was evaluated by flow cytometry using propidium iodide (PI) exclusion assay: 2 µg/mL of PI solution (Sigma-Aldrich, cat. P4864, St. Louis, MO, USA) was added to each sample and PI fluorescence was determined by FACS (CyAn ADP, Beckman Coulter, Fullerton, CA, USA).

    Techniques:

    α7nAChR expressed in THP-1 derived macrophages (TMs) inhibits the migration of CRC cells through the JAK2/STAT3 signaling pathway. TMs were treated with or without α-bungarotoxin (α-Btx) for 6 h and then with DMSO (control), AG490, LY294002 and Bay 11–7082 for 1 h, respectively. Subsequently, LoVo cells were co-cultured with TM in 24-well Transwell plates with 8 µm polycarbonate membrane filters for 36 h for the migration assay. (A) Upper panel: Representative images show the migration of LoVo cells under the condition that the TMs did not have the α-Btx treatment and were treated with AG490, LY294002 and Bay 11–7082 only, respectively. Lower panel: Representative images show the migration of LoVo cells under the condition that the TMs were treated with α-Btx only, or dually treated with AG490, LY294002 and Bay 11–7082, respectively. (B) Number of migrated LoVo cells are presented as the mean ± SD of six images (n=6) of three separate experiments. Significance of differences between groups was determined by Student's t-test. P-value

    Journal: Oncology Reports

    Article Title: α7 nicotinic acetylcholine receptor in tumor-associated macrophages inhibits colorectal cancer metastasis through the JAK2/STAT3 signaling pathway

    doi: 10.3892/or.2017.5935

    Figure Lengend Snippet: α7nAChR expressed in THP-1 derived macrophages (TMs) inhibits the migration of CRC cells through the JAK2/STAT3 signaling pathway. TMs were treated with or without α-bungarotoxin (α-Btx) for 6 h and then with DMSO (control), AG490, LY294002 and Bay 11–7082 for 1 h, respectively. Subsequently, LoVo cells were co-cultured with TM in 24-well Transwell plates with 8 µm polycarbonate membrane filters for 36 h for the migration assay. (A) Upper panel: Representative images show the migration of LoVo cells under the condition that the TMs did not have the α-Btx treatment and were treated with AG490, LY294002 and Bay 11–7082 only, respectively. Lower panel: Representative images show the migration of LoVo cells under the condition that the TMs were treated with α-Btx only, or dually treated with AG490, LY294002 and Bay 11–7082, respectively. (B) Number of migrated LoVo cells are presented as the mean ± SD of six images (n=6) of three separate experiments. Significance of differences between groups was determined by Student's t-test. P-value

    Article Snippet: To investigate the effects of signaling pathway inhibitors on TMα7−/− -enhanced migration of LoVo cells: TM cells (2×105 ) in 500 µl of medium in 24-well tissue culture plates were treated with or without α-bungarotoxin (α-Btx, a potent nicotinicα7 receptor antagonist; Sigma-Aldrich) at 100 nM for 6 h. After being washed with PBS three times, the TM cells were further treated with or without three signaling pathway inhibitors: AG490 (JAK2/STAT3 inhibitor, 20 µM), LY294002 (PI3K inhibitor, 5 µM) and Bay 11–7082 (NF-κB inhibitor, 2 µg/ml) for 1 h. AG490, LY294002, and Bay 11–7082 were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

    Techniques: Derivative Assay, Migration, Cell Culture

    The effect of STS on phosphorylated FOXO1, FOXO3A, and FOXO4 levels. Left: a representative Western-blot from left ventricle tissues isolated from each group showing the effect of STS on the levels of phosphorylated (p)-FOXO1, p-FOXO3A, p-FOXO4, total t-FOXO1, t-FOXO3A, and t-FOXO4. Right: the quantitative estimation of p-FOXO1, p-FOXO3A, and p-FOXO4 levels are shown as the relative OD values in STS and STS+LY294002 groups compared to the NS group. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    doi: 10.1038/aps.2013.91

    Figure Lengend Snippet: The effect of STS on phosphorylated FOXO1, FOXO3A, and FOXO4 levels. Left: a representative Western-blot from left ventricle tissues isolated from each group showing the effect of STS on the levels of phosphorylated (p)-FOXO1, p-FOXO3A, p-FOXO4, total t-FOXO1, t-FOXO3A, and t-FOXO4. Right: the quantitative estimation of p-FOXO1, p-FOXO3A, and p-FOXO4 levels are shown as the relative OD values in STS and STS+LY294002 groups compared to the NS group. b P

    Article Snippet: The inhibitor, LY294002, was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and dissolved in DMSO and PBS.

    Techniques: Western Blot, Isolation

    The effect of STS on Bim expression. (A) The expression levels of Bim mRNA (top) and protein (bottom) were analyzed using semi-quantitative RT-PCR and Western Blotting, respectively. The levels of GAPDH served as an internal control. The quantitative estimation of Bim mRNA and protein levels in the top right histogram are shown as the relative Bim mRNA and protein ratios in the STS and STS+LY294002 groups compared to the NS group. The expression levels of Bim mRNA and protein were measured in the myocardial tissue isolated from rats at 2 h after reperfusion. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    doi: 10.1038/aps.2013.91

    Figure Lengend Snippet: The effect of STS on Bim expression. (A) The expression levels of Bim mRNA (top) and protein (bottom) were analyzed using semi-quantitative RT-PCR and Western Blotting, respectively. The levels of GAPDH served as an internal control. The quantitative estimation of Bim mRNA and protein levels in the top right histogram are shown as the relative Bim mRNA and protein ratios in the STS and STS+LY294002 groups compared to the NS group. The expression levels of Bim mRNA and protein were measured in the myocardial tissue isolated from rats at 2 h after reperfusion. b P

    Article Snippet: The inhibitor, LY294002, was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and dissolved in DMSO and PBS.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Isolation

    The effect of STS on myocardial apoptosis after ischemia/reperfusion (I/R) injury. (A) Representative photomicrographs demonstrating TUNEL staining for apoptotic cells in the rat heart at 2 h after reperfusion in the NS, STS, and STS+LY294002 groups. The effects on the severity of cardiac apoptosis are shown in an average quantitative analysis of the rate of TUNEL-positive cells. The data are represented as the mean±SEM. n =8 for each group. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    doi: 10.1038/aps.2013.91

    Figure Lengend Snippet: The effect of STS on myocardial apoptosis after ischemia/reperfusion (I/R) injury. (A) Representative photomicrographs demonstrating TUNEL staining for apoptotic cells in the rat heart at 2 h after reperfusion in the NS, STS, and STS+LY294002 groups. The effects on the severity of cardiac apoptosis are shown in an average quantitative analysis of the rate of TUNEL-positive cells. The data are represented as the mean±SEM. n =8 for each group. b P

    Article Snippet: The inhibitor, LY294002, was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and dissolved in DMSO and PBS.

    Techniques: TUNEL Assay, Staining

    The H 2 O 2 -induced in vitro effect of STS on the apoptosis and caspase 3 activity of neonatal rat cardiac cells. (A) STS (3 μg/mL) was pretreated at 12 h before the incubation of cells with 200 μmol/L of H 2 O 2 for 1 h. Normal cells; control cells exposed to 200 μmol/L of H 2 O 2 for 1 h; 3 μg/mL STS-pretreated cells at 1 h after exposure to 200 μmol/L of H 2 O 2 (c); 3 μg/mL STS and 10 μmol/L LY294002 co-pretreated cells at 1 h after exposure to 200 μmol/L of H 2 O 2 (d). (B) Statistical analysis of the flow cytometric analysis data. (C) Statistical analysis of relative caspase 3 activity. The data represent the mean±SD. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    doi: 10.1038/aps.2013.91

    Figure Lengend Snippet: The H 2 O 2 -induced in vitro effect of STS on the apoptosis and caspase 3 activity of neonatal rat cardiac cells. (A) STS (3 μg/mL) was pretreated at 12 h before the incubation of cells with 200 μmol/L of H 2 O 2 for 1 h. Normal cells; control cells exposed to 200 μmol/L of H 2 O 2 for 1 h; 3 μg/mL STS-pretreated cells at 1 h after exposure to 200 μmol/L of H 2 O 2 (c); 3 μg/mL STS and 10 μmol/L LY294002 co-pretreated cells at 1 h after exposure to 200 μmol/L of H 2 O 2 (d). (B) Statistical analysis of the flow cytometric analysis data. (C) Statistical analysis of relative caspase 3 activity. The data represent the mean±SD. b P

    Article Snippet: The inhibitor, LY294002, was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and dissolved in DMSO and PBS.

    Techniques: In Vitro, Activity Assay, Incubation, Flow Cytometry

    The effect of STS on phosphorylated Akt levels. Top: representative WB from left ventricle tissue isolated from each group showing the effect of STS on the levels of phosphorylated (p)-Akt and total (t)-Akt. Bottom: the quantitative estimation of p-Akt levels is shown as the relative p-Akt and t-Akt ratios in STS and STS+LY294002 groups compared with the NS group. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    doi: 10.1038/aps.2013.91

    Figure Lengend Snippet: The effect of STS on phosphorylated Akt levels. Top: representative WB from left ventricle tissue isolated from each group showing the effect of STS on the levels of phosphorylated (p)-Akt and total (t)-Akt. Bottom: the quantitative estimation of p-Akt levels is shown as the relative p-Akt and t-Akt ratios in STS and STS+LY294002 groups compared with the NS group. b P

    Article Snippet: The inhibitor, LY294002, was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and dissolved in DMSO and PBS.

    Techniques: Western Blot, Isolation

    The effects of sodium tanshinone IIA sulfonate (STS) on infarct size, area at risk (AAR) (A), heart rate (HR) (B), the first derivative of developed pressure (d p /d t max ) (C), left ventricular developed pressure (LVDP) (D) and coronary flow (CF) (E). The isolated hearts from NS-, STS-, and STS+LY294002-treated rats were subjected to 30 min of global ischemia followed by 2 h of reperfusion. The results are expressed as the mean±SEM. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    doi: 10.1038/aps.2013.91

    Figure Lengend Snippet: The effects of sodium tanshinone IIA sulfonate (STS) on infarct size, area at risk (AAR) (A), heart rate (HR) (B), the first derivative of developed pressure (d p /d t max ) (C), left ventricular developed pressure (LVDP) (D) and coronary flow (CF) (E). The isolated hearts from NS-, STS-, and STS+LY294002-treated rats were subjected to 30 min of global ischemia followed by 2 h of reperfusion. The results are expressed as the mean±SEM. b P

    Article Snippet: The inhibitor, LY294002, was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and dissolved in DMSO and PBS.

    Techniques: Flow Cytometry, Isolation

    In vitro effect of STS on phospho-Akt, phospho-FOXO3A and Bim expression of neonatal rat cardiac cells induced by H 2 O 2 . (A) A representative Western blotting photograph showing the effect of STS on the levels of p-Akt, total-Akt, p-FOXO3A, and total-FOXO3A from each group. The quantitative estimation of phospho-Akt and phospho-FOXO3A levels was shown as the relative OD values in STS and STS+LY294002 groups compared with the normal and H 2 O 2 -induced control group. (B) The expression levels of Bim mRNA and protein were analyzed using a semi-quantitative RT-PCR and Western blotting, respectively. GAPDH served as an internal control. The quantitative estimation of Bim mRNA and protein levels in the histogram are shown as the relative Bim mRNA and protein ratios in the STS and STS+LY294002 groups compared to the normal and H 2 O 2 -induced control group.

    Journal: Acta Pharmacologica Sinica

    Article Title: Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    doi: 10.1038/aps.2013.91

    Figure Lengend Snippet: In vitro effect of STS on phospho-Akt, phospho-FOXO3A and Bim expression of neonatal rat cardiac cells induced by H 2 O 2 . (A) A representative Western blotting photograph showing the effect of STS on the levels of p-Akt, total-Akt, p-FOXO3A, and total-FOXO3A from each group. The quantitative estimation of phospho-Akt and phospho-FOXO3A levels was shown as the relative OD values in STS and STS+LY294002 groups compared with the normal and H 2 O 2 -induced control group. (B) The expression levels of Bim mRNA and protein were analyzed using a semi-quantitative RT-PCR and Western blotting, respectively. GAPDH served as an internal control. The quantitative estimation of Bim mRNA and protein levels in the histogram are shown as the relative Bim mRNA and protein ratios in the STS and STS+LY294002 groups compared to the normal and H 2 O 2 -induced control group.

    Article Snippet: The inhibitor, LY294002, was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and dissolved in DMSO and PBS.

    Techniques: In Vitro, Expressing, Western Blot, Quantitative RT-PCR

    RT-PCR analysis of GS-2, GBM8, and GBM6 cell lines treated with temozolomide, LY294002, and everolimus. CA-IX mRNA expression was significantly decreased following LY294002 treatment in each cell line: 10% ± 2% of control in GS-2 ( P = 2 ×

    Journal: Neuro-Oncology

    Article Title: Reduced phosphocholine and hyperpolarized lactate provide magnetic resonance biomarkers of PI3K/Akt/mTOR inhibition in glioblastoma

    doi: 10.1093/neuonc/nor209

    Figure Lengend Snippet: RT-PCR analysis of GS-2, GBM8, and GBM6 cell lines treated with temozolomide, LY294002, and everolimus. CA-IX mRNA expression was significantly decreased following LY294002 treatment in each cell line: 10% ± 2% of control in GS-2 ( P = 2 ×

    Article Snippet: For PI3K inhibition, cells were incubated with 50 µM LY294002 (LC Laboratories).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

    Western blot analysis of P-4EBP1, total-4EBP1, and CA-IX in GS-2, GBM6, and GBM8 cell lines treated with temozolomide, LY294002, and everolimus. In all 3 cell lines, P-4EBP1 and CA-IX protein levels were lower following treatment with everolimus and LY294002,

    Journal: Neuro-Oncology

    Article Title: Reduced phosphocholine and hyperpolarized lactate provide magnetic resonance biomarkers of PI3K/Akt/mTOR inhibition in glioblastoma

    doi: 10.1093/neuonc/nor209

    Figure Lengend Snippet: Western blot analysis of P-4EBP1, total-4EBP1, and CA-IX in GS-2, GBM6, and GBM8 cell lines treated with temozolomide, LY294002, and everolimus. In all 3 cell lines, P-4EBP1 and CA-IX protein levels were lower following treatment with everolimus and LY294002,

    Article Snippet: For PI3K inhibition, cells were incubated with 50 µM LY294002 (LC Laboratories).

    Techniques: Western Blot

    Western blot analysis of LDHA in GS-2, GBM8, and GBM6 cell lines treated with temozolomide, LY294002, and everolimus. In all 3 cell lines, LDHA protein levels were lower following treatment with everolimus and LY294002, in line with the reduced transcriptional

    Journal: Neuro-Oncology

    Article Title: Reduced phosphocholine and hyperpolarized lactate provide magnetic resonance biomarkers of PI3K/Akt/mTOR inhibition in glioblastoma

    doi: 10.1093/neuonc/nor209

    Figure Lengend Snippet: Western blot analysis of LDHA in GS-2, GBM8, and GBM6 cell lines treated with temozolomide, LY294002, and everolimus. In all 3 cell lines, LDHA protein levels were lower following treatment with everolimus and LY294002, in line with the reduced transcriptional

    Article Snippet: For PI3K inhibition, cells were incubated with 50 µM LY294002 (LC Laboratories).

    Techniques: Western Blot

    (A) PC level as measured by 1 H MRS in cell lysates as a function of time for control GS-2 cells and GS-2 cells treated with LY294002, everolimus, and temozolomide ( 3 repeats per treatment). The rate of PC production was 8.4 ± 0.2 fmol/cell/h

    Journal: Neuro-Oncology

    Article Title: Reduced phosphocholine and hyperpolarized lactate provide magnetic resonance biomarkers of PI3K/Akt/mTOR inhibition in glioblastoma

    doi: 10.1093/neuonc/nor209

    Figure Lengend Snippet: (A) PC level as measured by 1 H MRS in cell lysates as a function of time for control GS-2 cells and GS-2 cells treated with LY294002, everolimus, and temozolomide ( 3 repeats per treatment). The rate of PC production was 8.4 ± 0.2 fmol/cell/h

    Article Snippet: For PI3K inhibition, cells were incubated with 50 µM LY294002 (LC Laboratories).

    Techniques:

    Hyperpolarized lactate intensity as a function of time as assessed from HP 13 C MR spectra of control, temozolomide-treated, everolimus-treated, and LY294002-treated perfused GS-2 cells. Both LY294002 and everolimus induced a significant decrease in the

    Journal: Neuro-Oncology

    Article Title: Reduced phosphocholine and hyperpolarized lactate provide magnetic resonance biomarkers of PI3K/Akt/mTOR inhibition in glioblastoma

    doi: 10.1093/neuonc/nor209

    Figure Lengend Snippet: Hyperpolarized lactate intensity as a function of time as assessed from HP 13 C MR spectra of control, temozolomide-treated, everolimus-treated, and LY294002-treated perfused GS-2 cells. Both LY294002 and everolimus induced a significant decrease in the

    Article Snippet: For PI3K inhibition, cells were incubated with 50 µM LY294002 (LC Laboratories).

    Techniques:

    Modulation of PI3K-Akt pathway affects autophagic and phagocytic activity as well as inflammatory response in microglial cells. a A representative Western blot image of lysates of BV2 cells treated with LY294002 (LY) (0.1 µM – 10µM), and b with LY294002 together with LPS. c Quantification revealed that LY294002 decreased Akt S473 phosphorylation levels dose-dependently in cells treated with LY294002 and together with LPS and LY294002. d Quantification of LC3B II and I levels revealed a significant increase in LCB3II/I ratio by LY294002. e Phagocytic activity in BV2 cells was assessed using pHrodo bioparticles and fluorescence emission was measured in the IncuCyte live cell imaging device in every 15 min. BV2 cells were treated with four different concentrations of PI3K inhibitor LY294002 (5µM – 50µM) and with 5µM Cytochalasin D (CytD). LY294002 decreased phagocytic activity dose-dependently (p

    Journal: bioRxiv

    Article Title: Diabetic phenotype in mouse and humans with β-amyloid pathology reduces the number of microglia around β-amyloid plaques

    doi: 10.1101/2020.04.07.027292

    Figure Lengend Snippet: Modulation of PI3K-Akt pathway affects autophagic and phagocytic activity as well as inflammatory response in microglial cells. a A representative Western blot image of lysates of BV2 cells treated with LY294002 (LY) (0.1 µM – 10µM), and b with LY294002 together with LPS. c Quantification revealed that LY294002 decreased Akt S473 phosphorylation levels dose-dependently in cells treated with LY294002 and together with LPS and LY294002. d Quantification of LC3B II and I levels revealed a significant increase in LCB3II/I ratio by LY294002. e Phagocytic activity in BV2 cells was assessed using pHrodo bioparticles and fluorescence emission was measured in the IncuCyte live cell imaging device in every 15 min. BV2 cells were treated with four different concentrations of PI3K inhibitor LY294002 (5µM – 50µM) and with 5µM Cytochalasin D (CytD). LY294002 decreased phagocytic activity dose-dependently (p

    Article Snippet: After 1 h, PI3K inhibitor treatment with 1µM LY294002 (Life Technologies, Frederick, MD, USA) was started.

    Techniques: Activity Assay, Western Blot, Fluorescence, Live Cell Imaging

    Inhibition of PI3K activity affects phosphorylation of Syk in microglial cells. a Western blot analysis of lysates of BV2 cells pre-treated with LY294002 (LY, 1µM) or Src-kinase inhibitor SU6656 (SU, 2µM) prior to M-CSF treatment. b Quantification of Akt S473 phosphorylation levels revealed a significant decrease in LY294002-treated samples while total Akt levels were unchanged. c Quantification of Syk (Y525/526) phosphorylation levels revealed a significant increase in LY294002-treated samples while total Syk levels were unchanged. d Phosphorylation status of Syk was significantly increased in WT primary microglia treated with Trem2 antibody for 5 min as compared to control cells (ST). LY294002 treatment together with Trem2 antibody further increased phosphorylation. ST; starvation only, M-CSF; 100ng/ml for 5 min, LY; 1µM LY294002 for 1 h before M-CSF, SU; 2µM Src-kinase inhibitor SU6656 treatment 20 min before M-CSF. Data presented as mean % + SEM, n=3, *p

    Journal: bioRxiv

    Article Title: Diabetic phenotype in mouse and humans with β-amyloid pathology reduces the number of microglia around β-amyloid plaques

    doi: 10.1101/2020.04.07.027292

    Figure Lengend Snippet: Inhibition of PI3K activity affects phosphorylation of Syk in microglial cells. a Western blot analysis of lysates of BV2 cells pre-treated with LY294002 (LY, 1µM) or Src-kinase inhibitor SU6656 (SU, 2µM) prior to M-CSF treatment. b Quantification of Akt S473 phosphorylation levels revealed a significant decrease in LY294002-treated samples while total Akt levels were unchanged. c Quantification of Syk (Y525/526) phosphorylation levels revealed a significant increase in LY294002-treated samples while total Syk levels were unchanged. d Phosphorylation status of Syk was significantly increased in WT primary microglia treated with Trem2 antibody for 5 min as compared to control cells (ST). LY294002 treatment together with Trem2 antibody further increased phosphorylation. ST; starvation only, M-CSF; 100ng/ml for 5 min, LY; 1µM LY294002 for 1 h before M-CSF, SU; 2µM Src-kinase inhibitor SU6656 treatment 20 min before M-CSF. Data presented as mean % + SEM, n=3, *p

    Article Snippet: After 1 h, PI3K inhibitor treatment with 1µM LY294002 (Life Technologies, Frederick, MD, USA) was started.

    Techniques: Inhibition, Activity Assay, Western Blot

    PI3K signaling regulates B cell commitment and plasticity. ( a ) Cells from bm-derived wt and p110dKO pre-B cell lines were cultured either in the presence of IL-7, M-CSF, or without cytokines and analyzed by flow cytometry. Numbers indicate percentages of cells in the respective regions at day 7 after beginning of treatment. (Data are representative of at least 3 independent experiments). ( b ) FACS-analysis of CD11b expression of measured in cells from bm-derived wt and p110dKO pre-B cell lines cultured in the presence of IL-7 (upper panel). p110dKO cells were cultured in IL-7 or M-CSF supplemented medium and CD11b expression was assessed by FACS analysis at day 8 after beginning of treatment (lower panel). ( c ) Cells from a bm-derived SLP-65-deficient pre-B cell line were treated with LY294002 (30 μM) or solvent (DMSO) for 16 h and subsequently cultured in medium supplemented with M-CSF. Csf1r and Notch1 mRNA levels were determined with specific primers by qRT-PCR using the SYBR-Green detection method (left panel). Results are shown as mean ± SD of 2 independent analyses, run as duplicates. Statistical significance was calculated using the Mann-Whitney Test or the t-Test. Cells from the respective culture conditions were further analyzed by flow cytometry (right panel).

    Journal: Scientific Reports

    Article Title: PI3K induces B-cell development and regulates B cell identity

    doi: 10.1038/s41598-018-19460-5

    Figure Lengend Snippet: PI3K signaling regulates B cell commitment and plasticity. ( a ) Cells from bm-derived wt and p110dKO pre-B cell lines were cultured either in the presence of IL-7, M-CSF, or without cytokines and analyzed by flow cytometry. Numbers indicate percentages of cells in the respective regions at day 7 after beginning of treatment. (Data are representative of at least 3 independent experiments). ( b ) FACS-analysis of CD11b expression of measured in cells from bm-derived wt and p110dKO pre-B cell lines cultured in the presence of IL-7 (upper panel). p110dKO cells were cultured in IL-7 or M-CSF supplemented medium and CD11b expression was assessed by FACS analysis at day 8 after beginning of treatment (lower panel). ( c ) Cells from a bm-derived SLP-65-deficient pre-B cell line were treated with LY294002 (30 μM) or solvent (DMSO) for 16 h and subsequently cultured in medium supplemented with M-CSF. Csf1r and Notch1 mRNA levels were determined with specific primers by qRT-PCR using the SYBR-Green detection method (left panel). Results are shown as mean ± SD of 2 independent analyses, run as duplicates. Statistical significance was calculated using the Mann-Whitney Test or the t-Test. Cells from the respective culture conditions were further analyzed by flow cytometry (right panel).

    Article Snippet: For inhibition of PI3K, SLP-65-deficient pre-B cells and bone marrow derived wt pre-B cells were treated with 30 μM LY294002 (Merck Biosciences), Nalm-6 and SU-DHL-6 cells with 50 μM LY294002 for the indicated time points.

    Techniques: Derivative Assay, Cell Culture, Flow Cytometry, Cytometry, FACS, Expressing, Quantitative RT-PCR, SYBR Green Assay, MANN-WHITNEY

    Irf4 directly represses Pax5 expression. ( a ) Schematic overview showing the location of the Pax5 enhancer region within the Pax5 gene locus. ( b ) Irf-4 ChIP from cells of a SLP-65-deficient pre-B cell line. Amplified by qPCR was the enhancer region of Pax5 within exon 5 containing Irf4-binding sites (A and B). As control, amplification of a region 3.5 kb upstream of Pax5 enhancer was chosen. Both samples were normalized to mock control. ( c ) Schematic overview of the luciferase expression vector harboring a Vκ promoter and the respective Pax5 enhancer region containing or lacking the Irf4 binding sites. ( d–f ) WEHI cells were electroporated to introduce the empty vector (Vκ + Luciferase) or the indicated constructs containing or lacking the Irf4 binding sites. Expression of luciferase was equalized to the rLUC (Renilla) expression in each sample of co-transfected WEHI cells. In ( e ), WEHI cells were treated for 16 h with LY294002 upon electroporation and in ( f ), WEHI cells were co-transfected with either EV or with a vector encoding Irf4. Data are representative of at least 3 independent experiments and luciferase expression was determined using the Dual-Luciferase Reporter Assay System.

    Journal: Scientific Reports

    Article Title: PI3K induces B-cell development and regulates B cell identity

    doi: 10.1038/s41598-018-19460-5

    Figure Lengend Snippet: Irf4 directly represses Pax5 expression. ( a ) Schematic overview showing the location of the Pax5 enhancer region within the Pax5 gene locus. ( b ) Irf-4 ChIP from cells of a SLP-65-deficient pre-B cell line. Amplified by qPCR was the enhancer region of Pax5 within exon 5 containing Irf4-binding sites (A and B). As control, amplification of a region 3.5 kb upstream of Pax5 enhancer was chosen. Both samples were normalized to mock control. ( c ) Schematic overview of the luciferase expression vector harboring a Vκ promoter and the respective Pax5 enhancer region containing or lacking the Irf4 binding sites. ( d–f ) WEHI cells were electroporated to introduce the empty vector (Vκ + Luciferase) or the indicated constructs containing or lacking the Irf4 binding sites. Expression of luciferase was equalized to the rLUC (Renilla) expression in each sample of co-transfected WEHI cells. In ( e ), WEHI cells were treated for 16 h with LY294002 upon electroporation and in ( f ), WEHI cells were co-transfected with either EV or with a vector encoding Irf4. Data are representative of at least 3 independent experiments and luciferase expression was determined using the Dual-Luciferase Reporter Assay System.

    Article Snippet: For inhibition of PI3K, SLP-65-deficient pre-B cells and bone marrow derived wt pre-B cells were treated with 30 μM LY294002 (Merck Biosciences), Nalm-6 and SU-DHL-6 cells with 50 μM LY294002 for the indicated time points.

    Techniques: Expressing, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Luciferase, Plasmid Preparation, Introduce, Construct, Transfection, Electroporation, Reporter Assay

    PI3K regulates Pax5 expression. ( a ) Cells from a bone marrow (bm)-derived wildtype (wt) pre-B cell culture were transduced with constitutively active forms of AKT (myr-AKT), p110α (myr-p110α), or as control with empty vector (EV) and analyzed for pAKT and Pax5 expression by intracellular FACS. If not indicated otherwise, numbers in the histograms state the mean fluorescence intensity (MFI) of the respective GFP + populations. ( b ) Cells from a bm-derived wt pre-B cell culture were treated with LY294002 or DMSO for 16 h and analyzed for pAKT, Pax5, SLP-65 and FoxO1 expression by intracellular FACS. ( c ) Cells from a SLP-65-deficient pre-B cell line were treated with LY294002 or DMSO for 12 h and pAKT and Pax5 expression was analyzed by intracellular FACS. ( d ) Murine mature B cells (CD43 − ) were isolated and treated with LY294002 or DMSO for 12 h, lysed and subjected to immunoblot for analysis of Pax5 expression. Actin served as a loading control. For original full-length blots see Fig. S1e . ( e ) Total RNA of SLP-65-deficient cells treated for 12 h with LY294002 or DMSO was isolated. Gapdh and Pax5 mRNA-levels were detected with specific primers by qRT-PCR using the SYBR-Green detection method. Results are shown as mean ± SD of 2 independent experiments, run as duplicates. Statistical significance was calculated using the t-Test. ( f ) Total RNA of murine mature B cells treated with LY294002 or DMSO for 12 h was isolated. Gapdh and Pax5 mRNA-levels were detected with specific primers by qRT-PCR using the SYBR-Green detection method. Results are shown as mean ± SD of 2 independent experiments, run as duplicates. Statistical significance was calculated using the Mann-Whitney Test. Data shown in Fig. 1a–d are representative of at least 3 independent experiments.

    Journal: Scientific Reports

    Article Title: PI3K induces B-cell development and regulates B cell identity

    doi: 10.1038/s41598-018-19460-5

    Figure Lengend Snippet: PI3K regulates Pax5 expression. ( a ) Cells from a bone marrow (bm)-derived wildtype (wt) pre-B cell culture were transduced with constitutively active forms of AKT (myr-AKT), p110α (myr-p110α), or as control with empty vector (EV) and analyzed for pAKT and Pax5 expression by intracellular FACS. If not indicated otherwise, numbers in the histograms state the mean fluorescence intensity (MFI) of the respective GFP + populations. ( b ) Cells from a bm-derived wt pre-B cell culture were treated with LY294002 or DMSO for 16 h and analyzed for pAKT, Pax5, SLP-65 and FoxO1 expression by intracellular FACS. ( c ) Cells from a SLP-65-deficient pre-B cell line were treated with LY294002 or DMSO for 12 h and pAKT and Pax5 expression was analyzed by intracellular FACS. ( d ) Murine mature B cells (CD43 − ) were isolated and treated with LY294002 or DMSO for 12 h, lysed and subjected to immunoblot for analysis of Pax5 expression. Actin served as a loading control. For original full-length blots see Fig. S1e . ( e ) Total RNA of SLP-65-deficient cells treated for 12 h with LY294002 or DMSO was isolated. Gapdh and Pax5 mRNA-levels were detected with specific primers by qRT-PCR using the SYBR-Green detection method. Results are shown as mean ± SD of 2 independent experiments, run as duplicates. Statistical significance was calculated using the t-Test. ( f ) Total RNA of murine mature B cells treated with LY294002 or DMSO for 12 h was isolated. Gapdh and Pax5 mRNA-levels were detected with specific primers by qRT-PCR using the SYBR-Green detection method. Results are shown as mean ± SD of 2 independent experiments, run as duplicates. Statistical significance was calculated using the Mann-Whitney Test. Data shown in Fig. 1a–d are representative of at least 3 independent experiments.

    Article Snippet: For inhibition of PI3K, SLP-65-deficient pre-B cells and bone marrow derived wt pre-B cells were treated with 30 μM LY294002 (Merck Biosciences), Nalm-6 and SU-DHL-6 cells with 50 μM LY294002 for the indicated time points.

    Techniques: Expressing, Derivative Assay, Cell Culture, Transduction, Plasmid Preparation, FACS, Fluorescence, Isolation, Quantitative RT-PCR, SYBR Green Assay, MANN-WHITNEY

    FoxO1 induces Irf4 to repress Pax5. ( a ) Total RNA from a SLP-65-deficient pre-B cell line, treated with LY294002 or DMSO for 12 h, was isolated. Hprt and Irf4 mRNA-levels were detected with specific primers by qRT-PCR using the SYBR-Green detection method (n = 5). ( b ) Cells from a bm-derived wt pre-B cell line were transduced with FoxO1-A3 or EV and at day 1 post transduction total RNA was isolated. Expression of Hprt and Irf4 mRNA-levels were detected by qRT-PCR using a SYBR-Green detection method (n = 3). ( c ) Total RNA from p110dKO and bm-derived wt pre-B cell lines was analyzed for Hprt and Irf4 transcript levels by qRT-PCR using the SYBR-Green detection method (n = 3). ( d ) Hprt and Irf8 mRNA-levels were detected in RNA from Fig. 6a with specific primers by qRT-PCR using the SYBR-Green detection method (n = 5). ( e ) Similar to FoxO1-A3 in Fig. 5b , cells from a bm-derived wt pre-B cell line were transduced with Irf4 or EV and Pax5 expression was analyzed at day 2 after transduction by intracellular FACS. ( f ) Total RNA of cells from Fig. 6e was isolated to analyze transcript levels of Pax5 . Hprt and Pax5 mRNA-levels were detected by qRT-PCR using the SYBR-Green detection method (n = 3). ( g ) Human mature B cells from peripheral blood were isolated, treated with LY294002 and analyzed for IRF4 , PAX5 , BLNK and CD19 transcripts (n = 3). Results are shown as mean ± SD, run as duplicates. Data are representative of 2 ( c ) or at least 3 ( a,b , d–g ) independent experiments. Statistical significance was calculated using the t-Test.

    Journal: Scientific Reports

    Article Title: PI3K induces B-cell development and regulates B cell identity

    doi: 10.1038/s41598-018-19460-5

    Figure Lengend Snippet: FoxO1 induces Irf4 to repress Pax5. ( a ) Total RNA from a SLP-65-deficient pre-B cell line, treated with LY294002 or DMSO for 12 h, was isolated. Hprt and Irf4 mRNA-levels were detected with specific primers by qRT-PCR using the SYBR-Green detection method (n = 5). ( b ) Cells from a bm-derived wt pre-B cell line were transduced with FoxO1-A3 or EV and at day 1 post transduction total RNA was isolated. Expression of Hprt and Irf4 mRNA-levels were detected by qRT-PCR using a SYBR-Green detection method (n = 3). ( c ) Total RNA from p110dKO and bm-derived wt pre-B cell lines was analyzed for Hprt and Irf4 transcript levels by qRT-PCR using the SYBR-Green detection method (n = 3). ( d ) Hprt and Irf8 mRNA-levels were detected in RNA from Fig. 6a with specific primers by qRT-PCR using the SYBR-Green detection method (n = 5). ( e ) Similar to FoxO1-A3 in Fig. 5b , cells from a bm-derived wt pre-B cell line were transduced with Irf4 or EV and Pax5 expression was analyzed at day 2 after transduction by intracellular FACS. ( f ) Total RNA of cells from Fig. 6e was isolated to analyze transcript levels of Pax5 . Hprt and Pax5 mRNA-levels were detected by qRT-PCR using the SYBR-Green detection method (n = 3). ( g ) Human mature B cells from peripheral blood were isolated, treated with LY294002 and analyzed for IRF4 , PAX5 , BLNK and CD19 transcripts (n = 3). Results are shown as mean ± SD, run as duplicates. Data are representative of 2 ( c ) or at least 3 ( a,b , d–g ) independent experiments. Statistical significance was calculated using the t-Test.

    Article Snippet: For inhibition of PI3K, SLP-65-deficient pre-B cells and bone marrow derived wt pre-B cells were treated with 30 μM LY294002 (Merck Biosciences), Nalm-6 and SU-DHL-6 cells with 50 μM LY294002 for the indicated time points.

    Techniques: Isolation, Quantitative RT-PCR, SYBR Green Assay, Derivative Assay, Transduction, Expressing, FACS

    Inhibition of PI3K disrupts Akt/HIF-1 signaling and recreates liver IRI in Keap1HKO OLTs. Groups of WT or Keap1HKO liver donor mice were pre-treated with Ly294002 or DMSO (-1 h). (A) sALT levels (IU/L): (□) sham; ( ) WT+DMSO; ( ) WT+ Lly294002;

    Journal: Journal of hepatology

    Article Title: KEAP1-NRF2 COMPLEX IN ISCHEMIA-INDUCED HEPATOCELLULAR DAMAGE OF MOUSE LIVER TRANSPLANTS

    doi: 10.1016/j.jhep.2013.07.016

    Figure Lengend Snippet: Inhibition of PI3K disrupts Akt/HIF-1 signaling and recreates liver IRI in Keap1HKO OLTs. Groups of WT or Keap1HKO liver donor mice were pre-treated with Ly294002 or DMSO (-1 h). (A) sALT levels (IU/L): (□) sham; ( ) WT+DMSO; ( ) WT+ Lly294002;

    Article Snippet: In some experiments, donor mice were treated i.p. with PI3K inhibitor (LY294002; Calbiochem; 0.5mg/kg) or vehicle [10% dimethyl sulfoxide (DMSO) and 90% PBS] at 1h prior to liver procurement.

    Techniques: Inhibition, Mouse Assay

    NOX4 promotes NSCLC progression through activating PI3K/Akt pathway in vitro (A-C) NOX4 overexpressing A549 and H460 tumors-bearing animals were treated with LY294002 (25 mg/kg, every four days, i.p.).The growth curves of tumor (A), number of metastatic nodules on the surface of the lungs of mice (B), and Kaplan-Meier curves for illustration of the survival periods (C) were respectively shown. Bars are mean ± SD. *Significantly different from vector control, P

    Journal: Oncotarget

    Article Title: NOX4 promotes non-small cell lung cancer cell proliferation and metastasis through positive feedback regulation of PI3K/Akt signaling

    doi:

    Figure Lengend Snippet: NOX4 promotes NSCLC progression through activating PI3K/Akt pathway in vitro (A-C) NOX4 overexpressing A549 and H460 tumors-bearing animals were treated with LY294002 (25 mg/kg, every four days, i.p.).The growth curves of tumor (A), number of metastatic nodules on the surface of the lungs of mice (B), and Kaplan-Meier curves for illustration of the survival periods (C) were respectively shown. Bars are mean ± SD. *Significantly different from vector control, P

    Article Snippet: Materials Wartmannin and LY294002 (PI3K inhibitors) and PD98059 (MEK inhibitor) were obtained from Merck.

    Techniques: In Vitro, Mouse Assay, Plasmid Preparation

    NOX4 promotes NSCLC progression through activating PI3K/Akt pathway in vitro (A-C) Stably NOX4 overexpressing A549 and H460 cells were treated with 30 μM of LY294002 or 10 μM of Wortmannin and control solvent. The proliferation of cells was evaluated using MTT assay (A) and colony formation assay (B). The invasion of cells was evaluated using Matrigel transwell assay (C). (D) NOX4 overexpressing or silencing led to increase or decrease in Akt activation in A549 and H460 cells measured by western blotting. Akt activity was represented as the levels of phosphorylated or total forms of Akt. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P

    Journal: Oncotarget

    Article Title: NOX4 promotes non-small cell lung cancer cell proliferation and metastasis through positive feedback regulation of PI3K/Akt signaling

    doi:

    Figure Lengend Snippet: NOX4 promotes NSCLC progression through activating PI3K/Akt pathway in vitro (A-C) Stably NOX4 overexpressing A549 and H460 cells were treated with 30 μM of LY294002 or 10 μM of Wortmannin and control solvent. The proliferation of cells was evaluated using MTT assay (A) and colony formation assay (B). The invasion of cells was evaluated using Matrigel transwell assay (C). (D) NOX4 overexpressing or silencing led to increase or decrease in Akt activation in A549 and H460 cells measured by western blotting. Akt activity was represented as the levels of phosphorylated or total forms of Akt. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P

    Article Snippet: Materials Wartmannin and LY294002 (PI3K inhibitors) and PD98059 (MEK inhibitor) were obtained from Merck.

    Techniques: In Vitro, Stable Transfection, MTT Assay, Colony Assay, Transwell Assay, Activation Assay, Western Blot, Activity Assay, Plasmid Preparation

    PI3K/Akt pathway regulates NOX4 expression in NSCLC cells through NF-κB NOX4 interplays with PI3K/Akt pathway to regulate NSCLC progression. (A) Western blotting analysis of NOX4 expression in A549 and H460 cells treated with 30 μM LY294002 or 10 μM Wortmannin and control solvent for 24 hour. (B) Overexpression of Akt in A549 and H460 cells. Akt activity was represented as the levels of phosphorylated forms of Akt evaluated by western blotting. (C) NOX4 expression was evaluated in Akt overexpressing A549 and H460 cells using western blotting. (D) Western blotting analysis of NOX4 expression in AKT-overexpressing A549 and H460 cells after treated with 10 μM NF-κB inhibitor (Bay11-7082). (E) The ChIP assay was performed using the chromatin prepared from A549 and H460 cells. IgG served as the negative control. (F) 30 μM LY294002 and 10 μM Wortmannin inhibited the direct binding of NF-κB to the promoter region of NOX4 and NOX4 expression measured by ChIP assay in A549 and H460 cells.

    Journal: Oncotarget

    Article Title: NOX4 promotes non-small cell lung cancer cell proliferation and metastasis through positive feedback regulation of PI3K/Akt signaling

    doi:

    Figure Lengend Snippet: PI3K/Akt pathway regulates NOX4 expression in NSCLC cells through NF-κB NOX4 interplays with PI3K/Akt pathway to regulate NSCLC progression. (A) Western blotting analysis of NOX4 expression in A549 and H460 cells treated with 30 μM LY294002 or 10 μM Wortmannin and control solvent for 24 hour. (B) Overexpression of Akt in A549 and H460 cells. Akt activity was represented as the levels of phosphorylated forms of Akt evaluated by western blotting. (C) NOX4 expression was evaluated in Akt overexpressing A549 and H460 cells using western blotting. (D) Western blotting analysis of NOX4 expression in AKT-overexpressing A549 and H460 cells after treated with 10 μM NF-κB inhibitor (Bay11-7082). (E) The ChIP assay was performed using the chromatin prepared from A549 and H460 cells. IgG served as the negative control. (F) 30 μM LY294002 and 10 μM Wortmannin inhibited the direct binding of NF-κB to the promoter region of NOX4 and NOX4 expression measured by ChIP assay in A549 and H460 cells.

    Article Snippet: Materials Wartmannin and LY294002 (PI3K inhibitors) and PD98059 (MEK inhibitor) were obtained from Merck.

    Techniques: Expressing, Western Blot, Over Expression, Activity Assay, Chromatin Immunoprecipitation, Negative Control, Binding Assay

    Phospho-Akt and Akt expression, Phospho-GSK-3 β and GSK-3 β expression and Phospho-ERK1/2 and ERK1/2 expression in MIRI rats. ( A ) Phospho-Akt and Akt assay (1–4, represent results of Sham, MIRI, H-TFDM + MIRI + LY294002, H-TFDM + MIRI group, respectively, ## P

    Journal: Scientific Reports

    Article Title: Pretreatment with Total Flavonoid Extract from Dracocephalum Moldavica L. Attenuates Ischemia Reperfusion-induced Apoptosis

    doi: 10.1038/s41598-018-35726-4

    Figure Lengend Snippet: Phospho-Akt and Akt expression, Phospho-GSK-3 β and GSK-3 β expression and Phospho-ERK1/2 and ERK1/2 expression in MIRI rats. ( A ) Phospho-Akt and Akt assay (1–4, represent results of Sham, MIRI, H-TFDM + MIRI + LY294002, H-TFDM + MIRI group, respectively, ## P

    Article Snippet: LY294002 (PI3K inhibitor) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing

    BCP crystals induce expression of S100A8, S100A12, and MMP1 in primary macrophages in a Syk-, PI3K-, and ERK-dependent manner. Human macrophages were pre-treated with a piceatannol (50 μM, 100 μM), b LY294002 (25 μM, 50 μM), or c PD98059 (5 μM, 10 μM) for 30 mins prior to stimulation with BCP crystals for 24 h. mRNA levels of S100A8, S100A12, MMP1, and TIMP1 were analysed by real-time PCR. Results indicate mean (± SEM) of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

    Journal: Arthritis Research & Therapy

    Article Title: Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells

    doi: 10.1186/s13075-017-1225-0

    Figure Lengend Snippet: BCP crystals induce expression of S100A8, S100A12, and MMP1 in primary macrophages in a Syk-, PI3K-, and ERK-dependent manner. Human macrophages were pre-treated with a piceatannol (50 μM, 100 μM), b LY294002 (25 μM, 50 μM), or c PD98059 (5 μM, 10 μM) for 30 mins prior to stimulation with BCP crystals for 24 h. mRNA levels of S100A8, S100A12, MMP1, and TIMP1 were analysed by real-time PCR. Results indicate mean (± SEM) of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

    Article Snippet: Reagents Ultrapure lipopolysaccharide (LPS) and the PI3K inhibitor, LY294002, were from Invivogen (Toulouse, France).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Inhibition of Syk and PI3K reduces BCP crystal-induced IL-1 production in primary macrophages. Human macrophages (0.5 × 10 6 cells/well) were primed with LPS (100 ng/ml) for 2 h prior to treatment with a – c piceatannol (10 μM, 25 μM, and 50 μM) or d – f LY294002 (10 μM, 25 μM, and 50 μM) for 1 h and stimulation with BCP crystals (50 μg/ml) for 6 h. Cell supernatants were assessed for IL-1β, IL-1α, and TNF-α by ELISA. Results indicate mean (± SEM) of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs LPS + BCP

    Journal: Arthritis Research & Therapy

    Article Title: Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells

    doi: 10.1186/s13075-017-1225-0

    Figure Lengend Snippet: Inhibition of Syk and PI3K reduces BCP crystal-induced IL-1 production in primary macrophages. Human macrophages (0.5 × 10 6 cells/well) were primed with LPS (100 ng/ml) for 2 h prior to treatment with a – c piceatannol (10 μM, 25 μM, and 50 μM) or d – f LY294002 (10 μM, 25 μM, and 50 μM) for 1 h and stimulation with BCP crystals (50 μg/ml) for 6 h. Cell supernatants were assessed for IL-1β, IL-1α, and TNF-α by ELISA. Results indicate mean (± SEM) of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs LPS + BCP

    Article Snippet: Reagents Ultrapure lipopolysaccharide (LPS) and the PI3K inhibitor, LY294002, were from Invivogen (Toulouse, France).

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay

    BCP crystals activate MEK and ERK downstream of Syk/PI3K in primary macrophages and DC. a , b Human macrophages and c , d DC (2 × 10 6 cells/well) were stimulated with BCP crystals (50 μg/ml) for the indicated time points. Phosphorylation of MEK and ERK was detected by immunoblotting using phospho-specific antibodies. e , f Human macrophages and g , h DC were pre-treated with piceatannol (50 μM, 100 μM; lanes 3, 4), R788 (5 μM; lane 5), or LY294002 (50 μM; lane 6) for 30 min prior to stimulation with BCP crystals for 15 min. Phosphorylation of MEK and ERK was detected by immunoblotting. Representative blots of three independent experiments are shown. Densitometric analysis of three blots was performed using ImageJ software. Bar graphs illustrate the mean (± SEM) increase in phosphorylation, relative to the untreated sample (0) and normalised to total MEK/ERK protein. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

    Journal: Arthritis Research & Therapy

    Article Title: Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells

    doi: 10.1186/s13075-017-1225-0

    Figure Lengend Snippet: BCP crystals activate MEK and ERK downstream of Syk/PI3K in primary macrophages and DC. a , b Human macrophages and c , d DC (2 × 10 6 cells/well) were stimulated with BCP crystals (50 μg/ml) for the indicated time points. Phosphorylation of MEK and ERK was detected by immunoblotting using phospho-specific antibodies. e , f Human macrophages and g , h DC were pre-treated with piceatannol (50 μM, 100 μM; lanes 3, 4), R788 (5 μM; lane 5), or LY294002 (50 μM; lane 6) for 30 min prior to stimulation with BCP crystals for 15 min. Phosphorylation of MEK and ERK was detected by immunoblotting. Representative blots of three independent experiments are shown. Densitometric analysis of three blots was performed using ImageJ software. Bar graphs illustrate the mean (± SEM) increase in phosphorylation, relative to the untreated sample (0) and normalised to total MEK/ERK protein. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

    Article Snippet: Reagents Ultrapure lipopolysaccharide (LPS) and the PI3K inhibitor, LY294002, were from Invivogen (Toulouse, France).

    Techniques: Software