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    Autophagy inhibitor
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    94
    Thermo Fisher wortmannin
    The Arabidopsis U NFLATTENED AND S MALL L EAVES (USL1) regulates the function of PI3K by forming a complex with AtVPS30 and AtVPS34 (VPS, VACUOLAR PROTEIN SORTING). (a) Yeast two‐hybrid (Y2H) assays of USL1 with AtVPS30 and AtVPS34. NubWT represents the wild‐type (WT) N‐terminal half of ubiquitin. NubG represents the mutated N‐terminal half of ubiquitin. Transformed yeasts were spotted on control medium (‐2: SD‐Leu‐Trp) or selective medium (‐4: SD‐Leu‐Trp‐His‐Ade) at dilutions of 10‐, 100‐, and 1000‐fold. (b) The firefly luciferase (LUC) complementation imaging assays show that USL1 interacted with AtVPS30. LUC signals were detected in the combination of USL1‐nLUC and cLUC‐AtVPS30, but not in the control combinations including USL1‐nLUC and cLUC, nLUC and cLUC‐AtVPS30 and nLUC and cLUC. (c) The schematic representation of the USL1 deletions. (d) Y2H assays between differently truncated USL1 and AtVPS30. (e–g) The subcellular location of (e) the USL1‐GFP protein, (f) USL1ΔCC1‐GFP protein, and (g) USL1ΔCC2‐GFP protein. (h–m) The morphological changes of late endosome/multiple vesicle body/prevacuolar compartments (LE/MVB/PVC) following treatment with the PI3K inhibitor <t>Wortmannin.</t> The fluorescence of (h) USL1‐GFP, (i) RABF2a‐mCherry and (j) the merged picture after the mock treatment. (k) The fluorescence of USL1‐GFP, (l) RABF2a‐mCherry and (m) the merged picture after treatment with Wortmannin. GFP, green fluorescent protein. Bars, 10 μm.
    Wortmannin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 263 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore wortmannin
    Inhibition of autophagy enhanced the toxicity of 3,4,4′-THS in A549 cells. (A) A549 cells were treated with 3,4,4′-THS (40 μmol/L), 3-MA (5 mmol/L), <t>wortmannin</t> (2 μmol/L) or a combined treatment of 3,4,4′-THS and 3-MA or wortmannin for 3, 6, or 12 h. Microscopic photographs (400×) were taken under an inverted phase contrast microscope. (B) Cell viability determined by MTT assay. (C) Histogram shows the ratio of TUNEL-positive cells. (D) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. 3-MA was added to the cells for 1 h prior to 3,4,4′-THS treatment. (E) Histogram shows the relative level of cleaved PARP. (F) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. Wortmannin was added to the cells for 1 h prior to 3,4,4′-THS treatment. (G) Histogram shows the relative level of cleaved PARP. Data are expressed as the mean (±SEM) of three independent experiments. b P
    Wortmannin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc wortmannin
    Liraglutide stimulates PI3K‐dependent AKT signalling in cultured pancreatic βTC‐6 cells. Beta TC‐6 cells grown on 24‐well plates were pre‐treated or not with the PI3K inhibitor, <t>wortmannin</t> for 1 hr and finally stimulated with 1 nmol/l of liraglutide for 3–30 min. Cells were then lysed and AKT‐, BAD‐ and FoxO‐phosphorylation were determined by immunoblotting. Three independent experiments were performed. ( A ) Representative Western blot of (Ser473) AKT phosphorylation. Immunoblots were stripped and re‐probed with anti‐AKT antibody to normalize the blots for protein levels. ( B, C ) Representative Western blots of (Thr24) FoxO1/(Thr32) FoxO3a and (Ser136) BAD phosphorylation. Immunoblots were stripped and re‐probed with anti‐tubulin or anti‐Bad antibody to normalize the blots for protein levels.
    Wortmannin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 876 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Tocris wortmannin
    Liraglutide stimulates PI3K‐dependent AKT signalling in cultured pancreatic βTC‐6 cells. Beta TC‐6 cells grown on 24‐well plates were pre‐treated or not with the PI3K inhibitor, <t>wortmannin</t> for 1 hr and finally stimulated with 1 nmol/l of liraglutide for 3–30 min. Cells were then lysed and AKT‐, BAD‐ and FoxO‐phosphorylation were determined by immunoblotting. Three independent experiments were performed. ( A ) Representative Western blot of (Ser473) AKT phosphorylation. Immunoblots were stripped and re‐probed with anti‐AKT antibody to normalize the blots for protein levels. ( B, C ) Representative Western blots of (Thr24) FoxO1/(Thr32) FoxO3a and (Ser136) BAD phosphorylation. Immunoblots were stripped and re‐probed with anti‐tubulin or anti‐Bad antibody to normalize the blots for protein levels.
    Wortmannin, supplied by Tocris, used in various techniques. Bioz Stars score: 95/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals wortmannin
    Liraglutide stimulates PI3K‐dependent AKT signalling in cultured pancreatic βTC‐6 cells. Beta TC‐6 cells grown on 24‐well plates were pre‐treated or not with the PI3K inhibitor, <t>wortmannin</t> for 1 hr and finally stimulated with 1 nmol/l of liraglutide for 3–30 min. Cells were then lysed and AKT‐, BAD‐ and FoxO‐phosphorylation were determined by immunoblotting. Three independent experiments were performed. ( A ) Representative Western blot of (Ser473) AKT phosphorylation. Immunoblots were stripped and re‐probed with anti‐AKT antibody to normalize the blots for protein levels. ( B, C ) Representative Western blots of (Thr24) FoxO1/(Thr32) FoxO3a and (Ser136) BAD phosphorylation. Immunoblots were stripped and re‐probed with anti‐tubulin or anti‐Bad antibody to normalize the blots for protein levels.
    Wortmannin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH wortmannin
    BMS-345541 or <t>wortmannin</t> inhibited LPS-induced nuclear translocation of phospho p65 in chondrocytes as demonstrated by immunofluorescence microscopy . Primary human chondrocyte cultures either served as controls ( a-b : without primary antibody; c-d : with primary antibody) or were treated with lipopolysaccharides (LPS) alone (e-f) or were pre-treated with wortmannin (20 nM) (g-h) or with BMS-345541 (5 mM) (i-j) for 12 h before co-treatment with LPS (100 ng/ml) for 24 h before immunolabeling with phospho p65 antibodies and rhodamine-coupled secondary antibodies and counterstained with DAPI to visualize cell nuclei. Images shown are representative of three independent experiments. Magnification x400; bar, 30 nm.
    Wortmannin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem wortmannin
    Insulin-stimulated GLUT4 translocation in Drosophila fat body is inhibited by <t>wortmannin.</t> Confocal microscopy of HA-GLUT4-GFP-expression in fat body cells from animals reared on sugar-restricted diets. (A, B) Basal conditions. (C, D) After addition of 0.1 U/ml insulin; or (E, F) After pretreatment with 100 nM/L wortmannin, followed by insulin addition. GLUT4 was visualized in non-permeabilized cells by GFP fluorescence (green, A, C, E) or with anti-HA antibody (red B, D, F) to monitor membrane translocation Scale bar, 5 µm. (G) Surface exposure of HA-GLUT4 upon wortmannin/insulin treatment (G, light gray) or insulin only (G, dark gray). Values are expressed as pixel intensity of HA-GLUT4 in various conditions, as indicated. Fat bodies were collected from five animals each. Anti-HA labeling showed a significant inhibition of the insulin response in wortmannin treated samples (F, G) where fluorescence intensity (AU, arbitrary units) (1335±157; mean±SEM) was comparable to basal samples (1294±175) (B, G).
    Wortmannin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 210 article reviews
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    93
    Cayman Chemical wortmannin
    Insulin-stimulated GLUT4 translocation in Drosophila fat body is inhibited by <t>wortmannin.</t> Confocal microscopy of HA-GLUT4-GFP-expression in fat body cells from animals reared on sugar-restricted diets. (A, B) Basal conditions. (C, D) After addition of 0.1 U/ml insulin; or (E, F) After pretreatment with 100 nM/L wortmannin, followed by insulin addition. GLUT4 was visualized in non-permeabilized cells by GFP fluorescence (green, A, C, E) or with anti-HA antibody (red B, D, F) to monitor membrane translocation Scale bar, 5 µm. (G) Surface exposure of HA-GLUT4 upon wortmannin/insulin treatment (G, light gray) or insulin only (G, dark gray). Values are expressed as pixel intensity of HA-GLUT4 in various conditions, as indicated. Fat bodies were collected from five animals each. Anti-HA labeling showed a significant inhibition of the insulin response in wortmannin treated samples (F, G) where fluorescence intensity (AU, arbitrary units) (1335±157; mean±SEM) was comparable to basal samples (1294±175) (B, G).
    Wortmannin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories wortmannin
    The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins associated with the phosphoinositide 3-kinase (PI3K) signaling pathway in the rat glomerulus. ( a ) Staining of rat kidney sections with an antibody against GLCCI1 (red) is shown. The diabetic group showed low reactivity. However, GLCCI1 was regulated by <t>wortmannin</t> treatment. ( b – d ) The podocyte-specific proteins nephrin (green), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 following wortmannin treatment. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the diabetic group. We confirmed that SGK1 expression was decreased by wortmannin. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the control group. The lower reactivity of FOXO3A was accompanied by the overexpression of SGK1 in the diabetic group. The localization of FOXO3A was regulated by wortmannin treatment. Original magnification: × 400. Scale bar, 50 μm.
    Wortmannin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    InvivoGen wortmannin
    Activation of PI3K/Akt and MEK/ERK signaling pathways by direct interaction of PSaV in the absence of GCDCA. (A and B) LLC-PK cells were incubated with PSaV (MOI of 1 FFU/cell) in the absence of GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C and D) LLC-PK cells were mock pretreated or pretreated with <t>wortmannin</t> (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C (C) or transfected with or without siRNAs against PI3K p85α or MEK (D) and then infected with or without PSaV in the absence of GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (E and F) LLC-PK cells were incubated with PSaV VLPs (10 μg/ml), and the cell lysates were harvested at 5 mpi and prepared for Western blotting as described above. (G and H) LLC-PK cells were mock pretreated or pretreated with wortmannin or U0126 at the indicated doses for 1 h at 37°C (G) or transfected with or without siRNAs against PI3K p85α or MEK (H) and then incubated with or without PSaV VLPs. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.
    Wortmannin, supplied by InvivoGen, used in various techniques. Bioz Stars score: 97/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA wortmannin
    Local treatment with protein degradation inhibitors leads to dendritic MAPT mislocalization and spine loss. Rat hippocampal neurons (DIV 21–25) cultured in microfluidic chambers were treated on the neuritic side for 24 h either with DMSO (control, a), with the autophagy inhibitor <t>wortmannin</t> (b) or with the proteasomal inhibitor epoxomicin (c). MAP2 antibody (green), K9JA antibody (red) and DBN1 (cyan) were used to label dendrites, MAPT and spines, respectively. Only magnified images of the neuritic side are shown here. (a) In control, neurons treated with vehicle at the neuritic side (DMSO,
    Wortmannin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime wortmannin
    The 200-17-induced FAS and FASL phosphorylation was dependent on PI3K/AKT pathway activation in the LC cell lines. (a) AKT, phosphorylation of AKT, phosphorylated FOXO3, FAS, and FASL, as well as the gray intensity analysis in Hep-2 cells, stimulated with 200-17 for 0.5 h, 1 h, 4 h, 6 h, and 8 h, following pretreatment with <t>wortmannin</t> for 0.5 h. (b) AKT, phosphorylation of AKT, phosphorylated FOXO3, FAS, and FASL, as well as the gray intensity analysis in Tu212 cells, stimulated with 200-17 for 0.5 h, 1 h, 4 h, 6 h, and 8 h, following pretreatment with wortmannin for 0.5 h. n = 3 per group. One representative experiment of the three independent experiments is demonstrated. ∗ P
    Wortmannin, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM wortmannin
    Effects of <t>wortmannin</t> on TGF-β2-induced Smad-mut/Luc promoter activity in ARPE-19 cells. Transfected cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. Results are expressed relative to untreated control cells. Values represent the means ± SD of three independent experiments. * P
    Wortmannin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam wortmannin
    NF-κB activation by EVs relied on a dynamin-dependant endocytosis. (A) NF-κB activation by 1 µg.ml −1 EVs for 24 h in T84 cells pre-treated 2 h before with 20µM dynasore (N = 3). (B) NF-κB activation by 1 µg.ml −1 EV for 24 h in T84 cells pre-treated 2 h before with endocytosis inhibitors <t>wortmannin</t> 20 µM, genistein 50 µM, chlorpromazine 5 µM, pitstop2 20 µM, or Cdc42 inhibitor casin 10 µM (N ≥ 3). Data are expressed as median ± quartiles of fold change toward unstimulated cells.
    Wortmannin, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    17β hydroxy Wortmannin is an analog of wortmannin It irreversibly binds phosphoinositide 3 kinase PI3K and potently blocks fMLP stimulated respiratory burst in neutrophils IC 5 nM 17β hydroxy Wortmannin
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    N/A
    17β Hydroxywortmannin is an analog of wortmannin Cat No 1670 that irreversibly binds phosphoinositide 3 kinase PI3K and potently blocks fMLP stimulated respiratory burst in neutrophils IC₅₀ 5 nM 17β
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    N/A
    By phosphorylating phosphatidylinositol phosphoinositide 3 kinases PI3K activates diverse cellular functions including cell growth differentiation survival and motility Wortmannin is a potent cell permeable and irreversible inhibitor of PI3K enzymes
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    Image Search Results


    The Arabidopsis U NFLATTENED AND S MALL L EAVES (USL1) regulates the function of PI3K by forming a complex with AtVPS30 and AtVPS34 (VPS, VACUOLAR PROTEIN SORTING). (a) Yeast two‐hybrid (Y2H) assays of USL1 with AtVPS30 and AtVPS34. NubWT represents the wild‐type (WT) N‐terminal half of ubiquitin. NubG represents the mutated N‐terminal half of ubiquitin. Transformed yeasts were spotted on control medium (‐2: SD‐Leu‐Trp) or selective medium (‐4: SD‐Leu‐Trp‐His‐Ade) at dilutions of 10‐, 100‐, and 1000‐fold. (b) The firefly luciferase (LUC) complementation imaging assays show that USL1 interacted with AtVPS30. LUC signals were detected in the combination of USL1‐nLUC and cLUC‐AtVPS30, but not in the control combinations including USL1‐nLUC and cLUC, nLUC and cLUC‐AtVPS30 and nLUC and cLUC. (c) The schematic representation of the USL1 deletions. (d) Y2H assays between differently truncated USL1 and AtVPS30. (e–g) The subcellular location of (e) the USL1‐GFP protein, (f) USL1ΔCC1‐GFP protein, and (g) USL1ΔCC2‐GFP protein. (h–m) The morphological changes of late endosome/multiple vesicle body/prevacuolar compartments (LE/MVB/PVC) following treatment with the PI3K inhibitor Wortmannin. The fluorescence of (h) USL1‐GFP, (i) RABF2a‐mCherry and (j) the merged picture after the mock treatment. (k) The fluorescence of USL1‐GFP, (l) RABF2a‐mCherry and (m) the merged picture after treatment with Wortmannin. GFP, green fluorescent protein. Bars, 10 μm.

    Journal: The New Phytologist

    Article Title: The Arabidopsis USL1 controls multiple aspects of development by affecting late endosome morphology

    doi: 10.1111/nph.15249

    Figure Lengend Snippet: The Arabidopsis U NFLATTENED AND S MALL L EAVES (USL1) regulates the function of PI3K by forming a complex with AtVPS30 and AtVPS34 (VPS, VACUOLAR PROTEIN SORTING). (a) Yeast two‐hybrid (Y2H) assays of USL1 with AtVPS30 and AtVPS34. NubWT represents the wild‐type (WT) N‐terminal half of ubiquitin. NubG represents the mutated N‐terminal half of ubiquitin. Transformed yeasts were spotted on control medium (‐2: SD‐Leu‐Trp) or selective medium (‐4: SD‐Leu‐Trp‐His‐Ade) at dilutions of 10‐, 100‐, and 1000‐fold. (b) The firefly luciferase (LUC) complementation imaging assays show that USL1 interacted with AtVPS30. LUC signals were detected in the combination of USL1‐nLUC and cLUC‐AtVPS30, but not in the control combinations including USL1‐nLUC and cLUC, nLUC and cLUC‐AtVPS30 and nLUC and cLUC. (c) The schematic representation of the USL1 deletions. (d) Y2H assays between differently truncated USL1 and AtVPS30. (e–g) The subcellular location of (e) the USL1‐GFP protein, (f) USL1ΔCC1‐GFP protein, and (g) USL1ΔCC2‐GFP protein. (h–m) The morphological changes of late endosome/multiple vesicle body/prevacuolar compartments (LE/MVB/PVC) following treatment with the PI3K inhibitor Wortmannin. The fluorescence of (h) USL1‐GFP, (i) RABF2a‐mCherry and (j) the merged picture after the mock treatment. (k) The fluorescence of USL1‐GFP, (l) RABF2a‐mCherry and (m) the merged picture after treatment with Wortmannin. GFP, green fluorescent protein. Bars, 10 μm.

    Article Snippet: The seedlings were treated with 33 μM Wortmannin (Invitrogen, 3.3 mM stock in DMSO, diluted with deionized and distilled water) for 1 h, and the control was treated with the same concentration of DMSO diluted with deionized and distilled water.

    Techniques: Transformation Assay, Luciferase, Imaging, Fluorescence

    Inhibition of autophagy enhanced the toxicity of 3,4,4′-THS in A549 cells. (A) A549 cells were treated with 3,4,4′-THS (40 μmol/L), 3-MA (5 mmol/L), wortmannin (2 μmol/L) or a combined treatment of 3,4,4′-THS and 3-MA or wortmannin for 3, 6, or 12 h. Microscopic photographs (400×) were taken under an inverted phase contrast microscope. (B) Cell viability determined by MTT assay. (C) Histogram shows the ratio of TUNEL-positive cells. (D) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. 3-MA was added to the cells for 1 h prior to 3,4,4′-THS treatment. (E) Histogram shows the relative level of cleaved PARP. (F) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. Wortmannin was added to the cells for 1 h prior to 3,4,4′-THS treatment. (G) Histogram shows the relative level of cleaved PARP. Data are expressed as the mean (±SEM) of three independent experiments. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Resveratrol analogue 3,4,4′-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro

    doi: 10.1038/aps.2015.46

    Figure Lengend Snippet: Inhibition of autophagy enhanced the toxicity of 3,4,4′-THS in A549 cells. (A) A549 cells were treated with 3,4,4′-THS (40 μmol/L), 3-MA (5 mmol/L), wortmannin (2 μmol/L) or a combined treatment of 3,4,4′-THS and 3-MA or wortmannin for 3, 6, or 12 h. Microscopic photographs (400×) were taken under an inverted phase contrast microscope. (B) Cell viability determined by MTT assay. (C) Histogram shows the ratio of TUNEL-positive cells. (D) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. 3-MA was added to the cells for 1 h prior to 3,4,4′-THS treatment. (E) Histogram shows the relative level of cleaved PARP. (F) Western blot analysis of cleaved PARP in A549 cells treated without or with 3,4,4′-THS (40 μmol/L) for 3 h. Wortmannin was added to the cells for 1 h prior to 3,4,4′-THS treatment. (G) Histogram shows the relative level of cleaved PARP. Data are expressed as the mean (±SEM) of three independent experiments. b P

    Article Snippet: Wortmannin (Sigma-Aldrich, St Louis, MO, USA, W1628) was dissolved in DMSO.

    Techniques: Inhibition, Microscopy, MTT Assay, TUNEL Assay, Western Blot

    ROS was involved in 3,4,4′-THS induced autophagy and apoptosis in A549 cells. A549 cells were treated with 3,4,4′-THS (40 μmol/L), NAC (10 mmol/L) or a combined treatment of 3,4,4′-THS and NAC for 12 h. (A) Relative DCF fluorescence intensity, which corresponds to the ROS production level, measured with a microplate spectrophotometer. (B) Western blot analysis of LC3-II. (C) Histogram shows the relative level of LC3-II. (D) Cell viability determined by the MTT assay. (E) Histogram shows the ratio of TUNEL-positive cells. (F) Western blot analysis of cleaved PARP. (G) Histogram shows the relative level of cleaved PARP. (H) A549 cells were treated with 3,4,4′-THS (40 μmol/L), 3-MA (5 mmol/L), wortmannin (2 μmol/L) or a combined treatment of 3,4,4′-THS and 3-MA or Wortmannin for 12 h. The histogram shows the relative fluorescent intensity of DCF. Data are expressed as the mean (±SEM) of three independent experiments. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Resveratrol analogue 3,4,4′-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro

    doi: 10.1038/aps.2015.46

    Figure Lengend Snippet: ROS was involved in 3,4,4′-THS induced autophagy and apoptosis in A549 cells. A549 cells were treated with 3,4,4′-THS (40 μmol/L), NAC (10 mmol/L) or a combined treatment of 3,4,4′-THS and NAC for 12 h. (A) Relative DCF fluorescence intensity, which corresponds to the ROS production level, measured with a microplate spectrophotometer. (B) Western blot analysis of LC3-II. (C) Histogram shows the relative level of LC3-II. (D) Cell viability determined by the MTT assay. (E) Histogram shows the ratio of TUNEL-positive cells. (F) Western blot analysis of cleaved PARP. (G) Histogram shows the relative level of cleaved PARP. (H) A549 cells were treated with 3,4,4′-THS (40 μmol/L), 3-MA (5 mmol/L), wortmannin (2 μmol/L) or a combined treatment of 3,4,4′-THS and 3-MA or Wortmannin for 12 h. The histogram shows the relative fluorescent intensity of DCF. Data are expressed as the mean (±SEM) of three independent experiments. b P

    Article Snippet: Wortmannin (Sigma-Aldrich, St Louis, MO, USA, W1628) was dissolved in DMSO.

    Techniques: Fluorescence, Spectrophotometry, Western Blot, MTT Assay, TUNEL Assay

    COX-2 and EP4 mediated regulation of miR655. ( A ) COX-2 protein expression in MCF7, MCF7-Mock, MCF7-COX-2 and MCF7-miR655 cells. MiR655 expression in ( B ) MCF7-COX-2 and ( C ) SKBR3-COX-2 cells treated with COX-2 inhibitor (NS-398, 10 µM) and EP4 antagonist (ONO-AE3-208, 10 µM). ( D ) Comparison of fold changes in miR655 expression relative to controls (vehicle treatment) in a panel of breast cancer cell lines treated with an EP ligand (PGE2; 10 µM), EP4 receptor agonist (PGE1OH; 10 µM), or vehicle (DMSO). Comparison of miR655 expression after stimulation with PGE2 and PGE1OH followed by treatment with two PI3K inhibitors, Wortmannin (WT), LY-240-002 (LY) both at (10 µM) and ERK inhibitor U0126 (10 µM) in ( E ) MCF7 and in ( F ) T47D cells. To test intermediary role of NF-κB in EP4 mediated regulation of miR655 in ( G ) MCF7 and ( H ) T47D cells, the cells were treated with NF-κB inhibitor BAY-11-7082 (10 µM) or vehicle (DMSO) along with PGE2 and PGE1OH. Quantitative data are presented as means of triplicate experiments ± SEM. *Indicates p

    Journal: Scientific Reports

    Article Title: COX-2 induces oncogenic micro RNA miR655 in human breast cancer

    doi: 10.1038/s41598-017-18612-3

    Figure Lengend Snippet: COX-2 and EP4 mediated regulation of miR655. ( A ) COX-2 protein expression in MCF7, MCF7-Mock, MCF7-COX-2 and MCF7-miR655 cells. MiR655 expression in ( B ) MCF7-COX-2 and ( C ) SKBR3-COX-2 cells treated with COX-2 inhibitor (NS-398, 10 µM) and EP4 antagonist (ONO-AE3-208, 10 µM). ( D ) Comparison of fold changes in miR655 expression relative to controls (vehicle treatment) in a panel of breast cancer cell lines treated with an EP ligand (PGE2; 10 µM), EP4 receptor agonist (PGE1OH; 10 µM), or vehicle (DMSO). Comparison of miR655 expression after stimulation with PGE2 and PGE1OH followed by treatment with two PI3K inhibitors, Wortmannin (WT), LY-240-002 (LY) both at (10 µM) and ERK inhibitor U0126 (10 µM) in ( E ) MCF7 and in ( F ) T47D cells. To test intermediary role of NF-κB in EP4 mediated regulation of miR655 in ( G ) MCF7 and ( H ) T47D cells, the cells were treated with NF-κB inhibitor BAY-11-7082 (10 µM) or vehicle (DMSO) along with PGE2 and PGE1OH. Quantitative data are presented as means of triplicate experiments ± SEM. *Indicates p

    Article Snippet: Wortmannin (WM), an irreversible PI3K inhibitor, and LY-204002 (LY), a reversible PI3K inhibitor all purchased from Sigma-Aldrich, ERK1/2 inhibitor U0126 from Cell Signalling, MA.

    Techniques: Expressing

    Regulatory effect of RON160 and RON E5/6in on EMT-like activities in MDCK cells : A) Effect of RON, RON160, and RON E5/6in on epithelial/mesenchymal protein expression. Proteins (50 μg per sample) from cell lysates prepared after 72 h incubation were subjected to Western blot analysis using antibodies specific to vimentin and E-cadherin, respectively. Actin was used as the loading control. B ) Effect of RON, RON160 and RON E5/6in on cell morphological changes. MDCK, M-RON, M-RON160 and M-RON E5/6in cells were cultured for 24 h and then stimulated with 2 nM of MSP for 48 h. Cell morphological changes were observed under Olympus Inverted microscope and photographed. C ) Effect of RON, RON160 and RON E5/6in on spontaneous or MSP-induced MDCK cell migration. Cell monolayer was wounded as previously described [ 29 ] and stimulated with or without 2 nM of MSP for 16 h. Chemical inhibitors such as PD98059 (10 nM, specific to MAP kinase) and wortmannin (50 μg/ml, specific to PI-3 kinase) were added simultaneously. The wounded area covered by migrated cells was measured and shown as % of the covered space. Data shown here are from one of three experiments with similar results.

    Journal: Molecular Cancer

    Article Title: Deletion or insertion in the first immunoglobulin-plexin-transcription (IPT) domain differentially regulates expression and tumorigenic activities of RON receptor Tyrosine Kinase

    doi: 10.1186/1476-4598-9-307

    Figure Lengend Snippet: Regulatory effect of RON160 and RON E5/6in on EMT-like activities in MDCK cells : A) Effect of RON, RON160, and RON E5/6in on epithelial/mesenchymal protein expression. Proteins (50 μg per sample) from cell lysates prepared after 72 h incubation were subjected to Western blot analysis using antibodies specific to vimentin and E-cadherin, respectively. Actin was used as the loading control. B ) Effect of RON, RON160 and RON E5/6in on cell morphological changes. MDCK, M-RON, M-RON160 and M-RON E5/6in cells were cultured for 24 h and then stimulated with 2 nM of MSP for 48 h. Cell morphological changes were observed under Olympus Inverted microscope and photographed. C ) Effect of RON, RON160 and RON E5/6in on spontaneous or MSP-induced MDCK cell migration. Cell monolayer was wounded as previously described [ 29 ] and stimulated with or without 2 nM of MSP for 16 h. Chemical inhibitors such as PD98059 (10 nM, specific to MAP kinase) and wortmannin (50 μg/ml, specific to PI-3 kinase) were added simultaneously. The wounded area covered by migrated cells was measured and shown as % of the covered space. Data shown here are from one of three experiments with similar results.

    Article Snippet: PD98059 (PD), SB203580 (SB) and wortmannin (WT) were from Calbiochem (San Diego, CA).

    Techniques: Expressing, Incubation, Western Blot, Cell Culture, Inverted Microscopy, Migration

    HIF-1α induces cell growth mediated by the UCA1/PTEN/AKT signaling pathway. (A) HIF-1α-induced promotion of cell growth was abrogated by pPTEN or AKT inhibitor wortmannin in the MG-63 and U-2OS cells. (B) Western blot assays showed that HIF-1α-induced upregulation of Ki-67 was almost abolished by p-PTEN and wortmannin in the MG-63 and U-2OS cells. *P

    Journal: Oncology Reports

    Article Title: HIF-1α-induced upregulation of lncRNA UCA1 promotes cell growth in osteosarcoma by inactivating the PTEN/AKT signaling pathway

    doi: 10.3892/or.2018.6182

    Figure Lengend Snippet: HIF-1α induces cell growth mediated by the UCA1/PTEN/AKT signaling pathway. (A) HIF-1α-induced promotion of cell growth was abrogated by pPTEN or AKT inhibitor wortmannin in the MG-63 and U-2OS cells. (B) Western blot assays showed that HIF-1α-induced upregulation of Ki-67 was almost abolished by p-PTEN and wortmannin in the MG-63 and U-2OS cells. *P

    Article Snippet: The AKT inhibitor wortmannin was purchased from Sigma-Aldrich.

    Techniques: Western Blot

    lncRNA UCA1 regulates cell growth through inactivation of the PTEN/AKT signaling pathway. (A) Histogram shows the fold changes for the activities of different signaling pathways, as indicated by reporter activity. (B) Western blot experiments showed that lncRNA UCA1 suppressed PTEN expression and promoted p-AKT expression; however, no change in the total AKT (t-AKT) protein level was identified. (C) CCK-8 assay showed that transfection with p-PTEN or treatment with AKT inhibitor wortmannin potently abolished p-UCA1-induced promotion of cell growth in the osteosarcoma cells. (D) The p-UCA1-induced upregulation of Ki-67 was reversed by p-PTEN or wortmannin treatment. *P

    Journal: Oncology Reports

    Article Title: HIF-1α-induced upregulation of lncRNA UCA1 promotes cell growth in osteosarcoma by inactivating the PTEN/AKT signaling pathway

    doi: 10.3892/or.2018.6182

    Figure Lengend Snippet: lncRNA UCA1 regulates cell growth through inactivation of the PTEN/AKT signaling pathway. (A) Histogram shows the fold changes for the activities of different signaling pathways, as indicated by reporter activity. (B) Western blot experiments showed that lncRNA UCA1 suppressed PTEN expression and promoted p-AKT expression; however, no change in the total AKT (t-AKT) protein level was identified. (C) CCK-8 assay showed that transfection with p-PTEN or treatment with AKT inhibitor wortmannin potently abolished p-UCA1-induced promotion of cell growth in the osteosarcoma cells. (D) The p-UCA1-induced upregulation of Ki-67 was reversed by p-PTEN or wortmannin treatment. *P

    Article Snippet: The AKT inhibitor wortmannin was purchased from Sigma-Aldrich.

    Techniques: Activity Assay, Western Blot, Expressing, CCK-8 Assay, Transfection

    Liraglutide stimulates PI3K‐dependent AKT signalling in cultured pancreatic βTC‐6 cells. Beta TC‐6 cells grown on 24‐well plates were pre‐treated or not with the PI3K inhibitor, wortmannin for 1 hr and finally stimulated with 1 nmol/l of liraglutide for 3–30 min. Cells were then lysed and AKT‐, BAD‐ and FoxO‐phosphorylation were determined by immunoblotting. Three independent experiments were performed. ( A ) Representative Western blot of (Ser473) AKT phosphorylation. Immunoblots were stripped and re‐probed with anti‐AKT antibody to normalize the blots for protein levels. ( B, C ) Representative Western blots of (Thr24) FoxO1/(Thr32) FoxO3a and (Ser136) BAD phosphorylation. Immunoblots were stripped and re‐probed with anti‐tubulin or anti‐Bad antibody to normalize the blots for protein levels.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Liraglutide, a human glucagon‐like peptide‐1 analogue, stimulates AKT‐dependent survival signalling and inhibits pancreatic β‐cell apoptosis

    doi: 10.1111/jcmm.13259

    Figure Lengend Snippet: Liraglutide stimulates PI3K‐dependent AKT signalling in cultured pancreatic βTC‐6 cells. Beta TC‐6 cells grown on 24‐well plates were pre‐treated or not with the PI3K inhibitor, wortmannin for 1 hr and finally stimulated with 1 nmol/l of liraglutide for 3–30 min. Cells were then lysed and AKT‐, BAD‐ and FoxO‐phosphorylation were determined by immunoblotting. Three independent experiments were performed. ( A ) Representative Western blot of (Ser473) AKT phosphorylation. Immunoblots were stripped and re‐probed with anti‐AKT antibody to normalize the blots for protein levels. ( B, C ) Representative Western blots of (Thr24) FoxO1/(Thr32) FoxO3a and (Ser136) BAD phosphorylation. Immunoblots were stripped and re‐probed with anti‐tubulin or anti‐Bad antibody to normalize the blots for protein levels.

    Article Snippet: The PI3K inhibitor, wortmannin (#9951), was purchased from Cell Signaling.

    Techniques: Cell Culture, Western Blot

    APE1 and GFRα1 increases ERK phosphorylation in response to GDNF. ( A ) MIA PaCa-2 cells were pretreated with MEK-1 inhibitor PD98059 (10 μM) and the PI3K inhibitor Wortmannin (200 μM) and then treated with or without 50 ng/mL GDNF for 24 h. After treatment, cell proliferation was analyzed by WST-1 assay. Data are expressed as mean ± standard deviation from three independent experiments. ** and # denote p

    Journal: International Journal of Molecular Sciences

    Article Title: APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF

    doi: 10.3390/ijms21103586

    Figure Lengend Snippet: APE1 and GFRα1 increases ERK phosphorylation in response to GDNF. ( A ) MIA PaCa-2 cells were pretreated with MEK-1 inhibitor PD98059 (10 μM) and the PI3K inhibitor Wortmannin (200 μM) and then treated with or without 50 ng/mL GDNF for 24 h. After treatment, cell proliferation was analyzed by WST-1 assay. Data are expressed as mean ± standard deviation from three independent experiments. ** and # denote p

    Article Snippet: The MAPK/ERK kinase inhibitor PD98059 and the PI3K inhibitor Wortmannin were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: WST-1 Assay, Standard Deviation

    BMS-345541 or wortmannin inhibited LPS-induced nuclear translocation of phospho p65 in chondrocytes as demonstrated by immunofluorescence microscopy . Primary human chondrocyte cultures either served as controls ( a-b : without primary antibody; c-d : with primary antibody) or were treated with lipopolysaccharides (LPS) alone (e-f) or were pre-treated with wortmannin (20 nM) (g-h) or with BMS-345541 (5 mM) (i-j) for 12 h before co-treatment with LPS (100 ng/ml) for 24 h before immunolabeling with phospho p65 antibodies and rhodamine-coupled secondary antibodies and counterstained with DAPI to visualize cell nuclei. Images shown are representative of three independent experiments. Magnification x400; bar, 30 nm.

    Journal: Arthritis Research & Therapy

    Article Title: Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

    doi: 10.1186/ar4291

    Figure Lengend Snippet: BMS-345541 or wortmannin inhibited LPS-induced nuclear translocation of phospho p65 in chondrocytes as demonstrated by immunofluorescence microscopy . Primary human chondrocyte cultures either served as controls ( a-b : without primary antibody; c-d : with primary antibody) or were treated with lipopolysaccharides (LPS) alone (e-f) or were pre-treated with wortmannin (20 nM) (g-h) or with BMS-345541 (5 mM) (i-j) for 12 h before co-treatment with LPS (100 ng/ml) for 24 h before immunolabeling with phospho p65 antibodies and rhodamine-coupled secondary antibodies and counterstained with DAPI to visualize cell nuclei. Images shown are representative of three independent experiments. Magnification x400; bar, 30 nm.

    Article Snippet: Wortmannin was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Translocation Assay, Immunofluorescence, Microscopy, Immunolabeling

    LPS induced PI-3K/Akt activation and PI-3K inhibitor (wortmannin) suppressed this in a time- and dose-dependent manner . (A-B) Primary human chondrocytes in monolayer culture were either left untreated (controls), treated with lipopolysaccharides (LPS) (0, 0.1, 1, 10, 100 and 1000 ng/ml) for 50 min or stimulated with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blotting as described in Materials and methods. (C-D) Primary human chondrocytes in monolayer cultures were either left untreated or were prestimulated with wortmannin for the indicated concentrations and treated with 100 ng/ml LPS for 50 min, or prestimulated with 20 nM wortmannin for 12 h followed by co-treatment with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blot analysis as described in Materials and methods. Housekeeping protein β-actin served as a loading control and remained unaffected. PI-3K, phosphatidylinositol 3-kinase.

    Journal: Arthritis Research & Therapy

    Article Title: Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

    doi: 10.1186/ar4291

    Figure Lengend Snippet: LPS induced PI-3K/Akt activation and PI-3K inhibitor (wortmannin) suppressed this in a time- and dose-dependent manner . (A-B) Primary human chondrocytes in monolayer culture were either left untreated (controls), treated with lipopolysaccharides (LPS) (0, 0.1, 1, 10, 100 and 1000 ng/ml) for 50 min or stimulated with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blotting as described in Materials and methods. (C-D) Primary human chondrocytes in monolayer cultures were either left untreated or were prestimulated with wortmannin for the indicated concentrations and treated with 100 ng/ml LPS for 50 min, or prestimulated with 20 nM wortmannin for 12 h followed by co-treatment with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blot analysis as described in Materials and methods. Housekeeping protein β-actin served as a loading control and remained unaffected. PI-3K, phosphatidylinositol 3-kinase.

    Article Snippet: Wortmannin was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Activation Assay, Western Blot

    Inhibitory effects of BMS-345541 or/and wortmannin on LPS-induced NF-κB/PI-3K and apoptosis in primary human chondrocytes in vitro . Lipopolysaccharides (LPS)-induced disruption of cartilage may be induced through LPS complex formation with collagen II fibrils and through LPS/Toll-like receptor 4 (TLR4) association. Anti-collagen type II significantly reduced these procollagen-endotoxin complexes. Functional association with TLR4 leads to activation of intracellular downstream signaling pathway NF-κB, nuclear factor-κB (NF-κB)/PI-3K, phosphatidylinositol 3-kinase (PI-3K) inducing upregulation of matrix-degrading enzymes, inflammation and apoptosis. Culturing with specific inhibitors wortmannin (for PI-3K/AKT) and BMS-345541 (for IκB kinases (IKKs)) suppresses LPS-induced inflammatory response indicating the potential for new medical approaches.

    Journal: Arthritis Research & Therapy

    Article Title: Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

    doi: 10.1186/ar4291

    Figure Lengend Snippet: Inhibitory effects of BMS-345541 or/and wortmannin on LPS-induced NF-κB/PI-3K and apoptosis in primary human chondrocytes in vitro . Lipopolysaccharides (LPS)-induced disruption of cartilage may be induced through LPS complex formation with collagen II fibrils and through LPS/Toll-like receptor 4 (TLR4) association. Anti-collagen type II significantly reduced these procollagen-endotoxin complexes. Functional association with TLR4 leads to activation of intracellular downstream signaling pathway NF-κB, nuclear factor-κB (NF-κB)/PI-3K, phosphatidylinositol 3-kinase (PI-3K) inducing upregulation of matrix-degrading enzymes, inflammation and apoptosis. Culturing with specific inhibitors wortmannin (for PI-3K/AKT) and BMS-345541 (for IκB kinases (IKKs)) suppresses LPS-induced inflammatory response indicating the potential for new medical approaches.

    Article Snippet: Wortmannin was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: In Vitro, Functional Assay, Activation Assay

    LPS induced cellular and matrix degradation and apoptosis in cartilage tissue . Primary human chondrocytes were either left untreated (a) or were treated with 100 ng/ml lipopolysaccharides (LPS) (b-d) , pretreated with BMS-345541 (5 mM) (e-g) , wortmannin (20 nM) (h-j) followed by LPS treatment, or pretreated in combination with BMS-345541 and wortmannin (5 mM and 20 nM) (k-m) for 12 h and then stimulated with LPS for another 24 h. The cells were transferred to high-density culture for 14 days. Ultrastructural morphology was evaluated by electron microscopy. Control cultures of chondrocytes showed well-developed chondrocytes (Ch) embedded in a well-developed extracellular matrix (ECM) (a). Treatment with LPS resulted in matrix breakdown and cell lysis and apoptosis (arrows) (b-d). Pretreatment with BMS-345541 alone (e-g), with wortmannin alone (h-j) or in combination with BMS and wortmannin (k-m) resulted in a marked improvement of chondrocyte phenotype and the formation of cartilage nodules. The formation of a dense extracellular matrix (ECM) surrounding well-developed chondrocytes (Ch) was observed. Magnification x5000; bar, 1 µm.

    Journal: Arthritis Research & Therapy

    Article Title: Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

    doi: 10.1186/ar4291

    Figure Lengend Snippet: LPS induced cellular and matrix degradation and apoptosis in cartilage tissue . Primary human chondrocytes were either left untreated (a) or were treated with 100 ng/ml lipopolysaccharides (LPS) (b-d) , pretreated with BMS-345541 (5 mM) (e-g) , wortmannin (20 nM) (h-j) followed by LPS treatment, or pretreated in combination with BMS-345541 and wortmannin (5 mM and 20 nM) (k-m) for 12 h and then stimulated with LPS for another 24 h. The cells were transferred to high-density culture for 14 days. Ultrastructural morphology was evaluated by electron microscopy. Control cultures of chondrocytes showed well-developed chondrocytes (Ch) embedded in a well-developed extracellular matrix (ECM) (a). Treatment with LPS resulted in matrix breakdown and cell lysis and apoptosis (arrows) (b-d). Pretreatment with BMS-345541 alone (e-g), with wortmannin alone (h-j) or in combination with BMS and wortmannin (k-m) resulted in a marked improvement of chondrocyte phenotype and the formation of cartilage nodules. The formation of a dense extracellular matrix (ECM) surrounding well-developed chondrocytes (Ch) was observed. Magnification x5000; bar, 1 µm.

    Article Snippet: Wortmannin was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Electron Microscopy, Lysis

    Effects of BMS-345541 or/and wortmannin on the LPS-induced proinflammatory and apoptotic signaling in human chondrocytes . Primary human chondrocytes in monolayer cultures were either left untreated or stimulated with 100 ng/ml lipopolysaccharides (LPS), prestimulated with 5 mM BMS-345541, with 20 nM wortmannin or a combination of both inhibitors, BMS-345541 and wortmannin (5 mM and 20 nM) for 12 h and then treated with 100 ng/ml LPS for the indicated times. Whole-cell extracts were fractionated (500 ng protein per lane) on SDS-PAGE and examined by western blot analysis using anti-matrix metalloproteinase (MMP)-9 (A) , -MMP-13 ( B) , -cyclooxygenase-2 (Cox-2) (C) and anti-cleaved caspase-3 (D) . The results shown are representative of three independent experiments. Housekeeping protein β-actin served as a loading control.

    Journal: Arthritis Research & Therapy

    Article Title: Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

    doi: 10.1186/ar4291

    Figure Lengend Snippet: Effects of BMS-345541 or/and wortmannin on the LPS-induced proinflammatory and apoptotic signaling in human chondrocytes . Primary human chondrocytes in monolayer cultures were either left untreated or stimulated with 100 ng/ml lipopolysaccharides (LPS), prestimulated with 5 mM BMS-345541, with 20 nM wortmannin or a combination of both inhibitors, BMS-345541 and wortmannin (5 mM and 20 nM) for 12 h and then treated with 100 ng/ml LPS for the indicated times. Whole-cell extracts were fractionated (500 ng protein per lane) on SDS-PAGE and examined by western blot analysis using anti-matrix metalloproteinase (MMP)-9 (A) , -MMP-13 ( B) , -cyclooxygenase-2 (Cox-2) (C) and anti-cleaved caspase-3 (D) . The results shown are representative of three independent experiments. Housekeeping protein β-actin served as a loading control.

    Article Snippet: Wortmannin was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: SDS Page, Western Blot

    LPS induced the phosphorylation and translocation of p65 and the IKK inhibitor BMS-345541 or PI-3K inhibitor wortmannin suppressed this in a time- and dose-dependent manner . (A-B) Primary human chondrocytes in monolayer culture were either left untreated (controls), treated with lipopolysaccharides (LPS) (0, 0.1, 1, 10, 100 and 1000 ng/ml) for 30 min (A) or stimulated with 100 ng/ml LPS for the indicated times (B). Nuclear extracts were prepared and assayed for nuclear factor-κB (NF-κB) (p65) activation by western blot analysis as described in Materials and methods. (C-F) Primary human chondrocytes in monolayer cultures were either left alone or pretreated with BMS-345541 or with wortmannin for the indicated concentrations followed by treatment with 100 ng/ml LPS for 30 min (C and E), or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h and co-treated with 100 ng/ml LPS for the indicated times (D and F). Nuclear extracts were prepared and assayed for NF-κB (p65) activation by western blot analysis as described in Materials and methods. Synthesis of poly(ADP-ribose) polymerase (PARP) remained unaffected in nuclear extracts. IKK, IκB kinase; PI-3K, phosphatidylinositol 3-kinase.

    Journal: Arthritis Research & Therapy

    Article Title: Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

    doi: 10.1186/ar4291

    Figure Lengend Snippet: LPS induced the phosphorylation and translocation of p65 and the IKK inhibitor BMS-345541 or PI-3K inhibitor wortmannin suppressed this in a time- and dose-dependent manner . (A-B) Primary human chondrocytes in monolayer culture were either left untreated (controls), treated with lipopolysaccharides (LPS) (0, 0.1, 1, 10, 100 and 1000 ng/ml) for 30 min (A) or stimulated with 100 ng/ml LPS for the indicated times (B). Nuclear extracts were prepared and assayed for nuclear factor-κB (NF-κB) (p65) activation by western blot analysis as described in Materials and methods. (C-F) Primary human chondrocytes in monolayer cultures were either left alone or pretreated with BMS-345541 or with wortmannin for the indicated concentrations followed by treatment with 100 ng/ml LPS for 30 min (C and E), or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h and co-treated with 100 ng/ml LPS for the indicated times (D and F). Nuclear extracts were prepared and assayed for NF-κB (p65) activation by western blot analysis as described in Materials and methods. Synthesis of poly(ADP-ribose) polymerase (PARP) remained unaffected in nuclear extracts. IKK, IκB kinase; PI-3K, phosphatidylinositol 3-kinase.

    Article Snippet: Wortmannin was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Translocation Assay, Activation Assay, Western Blot

    Effects of BMS-345541 and wortmannin on LPS signalling in chondrocytes . (A) Effects of BMS-345541 or wortmannin on LPS-induced IκB-α phosphorylation and degradation in chondrocytes in monolayer cultures . Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml lipopolysaccharides (LPS) or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by LPS treatment (100 ng/ml) for the indicated times. Cytoplasmic extracts were prepared, fractionated (500 ng protein per lane) on 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes. Western blot analysis was performed with anti-phospho-specific-IκB-α (II), anti-IκB-α (I) and anti-β-actin (II, control). The results shown are representative of three independent experiments. (B) Effects of BMS-345541 or wortmannin on LPS-induced IKK activation in chondrocytes in monolayer cultures . Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml LPS or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by treatment with 100 ng/ml LPS for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IkB kinase (IKK) and then analyzed by an immune complex kinase assay as described in Materials and methods. To examine the effect of LPS, BMS-345541 or wortmannin on the level of activation of IKK proteins, whole-cell extracts were fractionated (500 ng protein per lane) on SDS-PAGE and examined by western blot analysis using anti-phospho-specific-IκB-α and anti-IκB-β (I). LPS, BMS-345541 or wortmannin had no direct effect on the expression of IKK-α or IKK-β proteins (II, III). The results shown are representative of three independent experiments.

    Journal: Arthritis Research & Therapy

    Article Title: Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

    doi: 10.1186/ar4291

    Figure Lengend Snippet: Effects of BMS-345541 and wortmannin on LPS signalling in chondrocytes . (A) Effects of BMS-345541 or wortmannin on LPS-induced IκB-α phosphorylation and degradation in chondrocytes in monolayer cultures . Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml lipopolysaccharides (LPS) or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by LPS treatment (100 ng/ml) for the indicated times. Cytoplasmic extracts were prepared, fractionated (500 ng protein per lane) on 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes. Western blot analysis was performed with anti-phospho-specific-IκB-α (II), anti-IκB-α (I) and anti-β-actin (II, control). The results shown are representative of three independent experiments. (B) Effects of BMS-345541 or wortmannin on LPS-induced IKK activation in chondrocytes in monolayer cultures . Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml LPS or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by treatment with 100 ng/ml LPS for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IkB kinase (IKK) and then analyzed by an immune complex kinase assay as described in Materials and methods. To examine the effect of LPS, BMS-345541 or wortmannin on the level of activation of IKK proteins, whole-cell extracts were fractionated (500 ng protein per lane) on SDS-PAGE and examined by western blot analysis using anti-phospho-specific-IκB-α and anti-IκB-β (I). LPS, BMS-345541 or wortmannin had no direct effect on the expression of IKK-α or IKK-β proteins (II, III). The results shown are representative of three independent experiments.

    Article Snippet: Wortmannin was purchased from Biomol (Plymouth Meeting, PA, USA).

    Techniques: SDS Page, Western Blot, Activation Assay, Immunoprecipitation, Immune Complex Kinase Assay, Expressing

    Insulin-stimulated GLUT4 translocation in Drosophila fat body is inhibited by wortmannin. Confocal microscopy of HA-GLUT4-GFP-expression in fat body cells from animals reared on sugar-restricted diets. (A, B) Basal conditions. (C, D) After addition of 0.1 U/ml insulin; or (E, F) After pretreatment with 100 nM/L wortmannin, followed by insulin addition. GLUT4 was visualized in non-permeabilized cells by GFP fluorescence (green, A, C, E) or with anti-HA antibody (red B, D, F) to monitor membrane translocation Scale bar, 5 µm. (G) Surface exposure of HA-GLUT4 upon wortmannin/insulin treatment (G, light gray) or insulin only (G, dark gray). Values are expressed as pixel intensity of HA-GLUT4 in various conditions, as indicated. Fat bodies were collected from five animals each. Anti-HA labeling showed a significant inhibition of the insulin response in wortmannin treated samples (F, G) where fluorescence intensity (AU, arbitrary units) (1335±157; mean±SEM) was comparable to basal samples (1294±175) (B, G).

    Journal: PLoS ONE

    Article Title: Insulin Stimulates Translocation of Human GLUT4 to the Membrane in Fat Bodies of Transgenic Drosophila melanogaster

    doi: 10.1371/journal.pone.0077953

    Figure Lengend Snippet: Insulin-stimulated GLUT4 translocation in Drosophila fat body is inhibited by wortmannin. Confocal microscopy of HA-GLUT4-GFP-expression in fat body cells from animals reared on sugar-restricted diets. (A, B) Basal conditions. (C, D) After addition of 0.1 U/ml insulin; or (E, F) After pretreatment with 100 nM/L wortmannin, followed by insulin addition. GLUT4 was visualized in non-permeabilized cells by GFP fluorescence (green, A, C, E) or with anti-HA antibody (red B, D, F) to monitor membrane translocation Scale bar, 5 µm. (G) Surface exposure of HA-GLUT4 upon wortmannin/insulin treatment (G, light gray) or insulin only (G, dark gray). Values are expressed as pixel intensity of HA-GLUT4 in various conditions, as indicated. Fat bodies were collected from five animals each. Anti-HA labeling showed a significant inhibition of the insulin response in wortmannin treated samples (F, G) where fluorescence intensity (AU, arbitrary units) (1335±157; mean±SEM) was comparable to basal samples (1294±175) (B, G).

    Article Snippet: For testing effects of wortmannin on HA-GLUT4-GFP translocation, fat bodies from animals fed on a sugar-restricted diet were isolated and pre-incubated in RPMI for 20 min with 100 nM/L wortmannin (Enzo Life Sciences Inc.) at RT prior to insulin addition.

    Techniques: Translocation Assay, Confocal Microscopy, Expressing, Fluorescence, Labeling, Inhibition

    The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins associated with the phosphoinositide 3-kinase (PI3K) signaling pathway in the rat glomerulus. ( a ) Staining of rat kidney sections with an antibody against GLCCI1 (red) is shown. The diabetic group showed low reactivity. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 following wortmannin treatment. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the diabetic group. We confirmed that SGK1 expression was decreased by wortmannin. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the control group. The lower reactivity of FOXO3A was accompanied by the overexpression of SGK1 in the diabetic group. The localization of FOXO3A was regulated by wortmannin treatment. Original magnification: × 400. Scale bar, 50 μm.

    Journal: Experimental & Molecular Medicine

    Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    doi: 10.1038/emm.2016.28

    Figure Lengend Snippet: The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins associated with the phosphoinositide 3-kinase (PI3K) signaling pathway in the rat glomerulus. ( a ) Staining of rat kidney sections with an antibody against GLCCI1 (red) is shown. The diabetic group showed low reactivity. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 following wortmannin treatment. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the diabetic group. We confirmed that SGK1 expression was decreased by wortmannin. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the control group. The lower reactivity of FOXO3A was accompanied by the overexpression of SGK1 in the diabetic group. The localization of FOXO3A was regulated by wortmannin treatment. Original magnification: × 400. Scale bar, 50 μm.

    Article Snippet: Podocytes from the three groups were incubated in culture medium containing either 5 or 25 mM glucose for 24 h. After 24 h of incubation, the wortmannin group incubated with high glucose was treated with wortmannin (0.32 μM , LC Laboratories, Woburn, MA, USA); the half-maximal inhibitory concentration of wortmannin was determined by CCK-8 assay (data not shown).

    Techniques: Staining, Expressing, Over Expression

    Colocalization of glucocorticoid-induced transcript 1 (GLCCI1) with the podocyte-specific proteins nephrin and synaptopodin. ( a ) Double labeling with the podocyte-specific marker nephrin (green) showed a complete overlap (merge, yellow) with the distribution of GLCCI1 (red) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( b ) GLCCI1 (red) also colocalized with synaptopodin (green). We observed overlapping reactivity (yellow indicates red plus green) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( c , d ) In rat glomeruli, we observed overlapping reactivity (yellow) via the colocalization of GLCCI1 (red) with nephrin and synaptopodin (green). Wortmannin ameliorated the lower reactivity in glomeruli from rats in the diabetic group. Original magnification: × 400. Scale bar, 50 μm.

    Journal: Experimental & Molecular Medicine

    Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    doi: 10.1038/emm.2016.28

    Figure Lengend Snippet: Colocalization of glucocorticoid-induced transcript 1 (GLCCI1) with the podocyte-specific proteins nephrin and synaptopodin. ( a ) Double labeling with the podocyte-specific marker nephrin (green) showed a complete overlap (merge, yellow) with the distribution of GLCCI1 (red) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( b ) GLCCI1 (red) also colocalized with synaptopodin (green). We observed overlapping reactivity (yellow indicates red plus green) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( c , d ) In rat glomeruli, we observed overlapping reactivity (yellow) via the colocalization of GLCCI1 (red) with nephrin and synaptopodin (green). Wortmannin ameliorated the lower reactivity in glomeruli from rats in the diabetic group. Original magnification: × 400. Scale bar, 50 μm.

    Article Snippet: Podocytes from the three groups were incubated in culture medium containing either 5 or 25 mM glucose for 24 h. After 24 h of incubation, the wortmannin group incubated with high glucose was treated with wortmannin (0.32 μM , LC Laboratories, Woburn, MA, USA); the half-maximal inhibitory concentration of wortmannin was determined by CCK-8 assay (data not shown).

    Techniques: Labeling, Marker, Cell Culture

    Analysis of glucocorticoid-induced transcript 1 (GLCCI1) protein expression and involvement in the phosphoinositide 3-kinase (PI3K) pathway by western blotting. ( a ) GLCCI1, nephrin and podocin were expressed only in the 5 m M and the wortmannin-treated diabetic groups. Serum/glucocorticoid-regulated kinase 1 (SGK1) was expressed only in the high glucose-induced group. Forkhead box O3 (FOXO3A) was regulated by wortmannin. ( b ) GLCCI1 was expressed only in the control and wortmannin-treated diabetic groups. GLCCI1 was significantly decreased in the diabetic group. ( c , d ) The relative band intensities of proteins in the podocytes and the kidneys of diabetic rats were observed by western blot analysis. The band intensities were measured using the Multi Gauge V3.0 software (Fuji Film). *** P

    Journal: Experimental & Molecular Medicine

    Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    doi: 10.1038/emm.2016.28

    Figure Lengend Snippet: Analysis of glucocorticoid-induced transcript 1 (GLCCI1) protein expression and involvement in the phosphoinositide 3-kinase (PI3K) pathway by western blotting. ( a ) GLCCI1, nephrin and podocin were expressed only in the 5 m M and the wortmannin-treated diabetic groups. Serum/glucocorticoid-regulated kinase 1 (SGK1) was expressed only in the high glucose-induced group. Forkhead box O3 (FOXO3A) was regulated by wortmannin. ( b ) GLCCI1 was expressed only in the control and wortmannin-treated diabetic groups. GLCCI1 was significantly decreased in the diabetic group. ( c , d ) The relative band intensities of proteins in the podocytes and the kidneys of diabetic rats were observed by western blot analysis. The band intensities were measured using the Multi Gauge V3.0 software (Fuji Film). *** P

    Article Snippet: Podocytes from the three groups were incubated in culture medium containing either 5 or 25 mM glucose for 24 h. After 24 h of incubation, the wortmannin group incubated with high glucose was treated with wortmannin (0.32 μM , LC Laboratories, Woburn, MA, USA); the half-maximal inhibitory concentration of wortmannin was determined by CCK-8 assay (data not shown).

    Techniques: Expressing, Western Blot, Software

    The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins involved in the phosphoinositide 3-kinase (PI3K) signaling pathway in podocytes. ( a ) In podocytes, GLCCI1 (red, Alexa 568 conjugated) was localized in the 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei. No GLCCI1 localization was observed in the 25 m M D -glucose-treated podocytes. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green, Alexa 488 conjugated), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 in podocytes treated with wortmannin. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the 25 m M group. We confirmed that treatment with wortmannin decreased SGK1 expression. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the 5 m M group. A lower FOXO3A signal was accompanied by high reactivity of SGK1 in the 25 m M group. Localization of FOXO3A was regulated by treatment with wortmannin. Original magnification: × 400. Scale bar, 50 μm.

    Journal: Experimental & Molecular Medicine

    Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    doi: 10.1038/emm.2016.28

    Figure Lengend Snippet: The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins involved in the phosphoinositide 3-kinase (PI3K) signaling pathway in podocytes. ( a ) In podocytes, GLCCI1 (red, Alexa 568 conjugated) was localized in the 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei. No GLCCI1 localization was observed in the 25 m M D -glucose-treated podocytes. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green, Alexa 488 conjugated), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 in podocytes treated with wortmannin. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the 25 m M group. We confirmed that treatment with wortmannin decreased SGK1 expression. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the 5 m M group. A lower FOXO3A signal was accompanied by high reactivity of SGK1 in the 25 m M group. Localization of FOXO3A was regulated by treatment with wortmannin. Original magnification: × 400. Scale bar, 50 μm.

    Article Snippet: Podocytes from the three groups were incubated in culture medium containing either 5 or 25 mM glucose for 24 h. After 24 h of incubation, the wortmannin group incubated with high glucose was treated with wortmannin (0.32 μM , LC Laboratories, Woburn, MA, USA); the half-maximal inhibitory concentration of wortmannin was determined by CCK-8 assay (data not shown).

    Techniques: Staining, Expressing

    Detection of glucocorticoid-induced transcript 1 (GLCCI1) and podocyte-specific marker gene expression by reverse transcription-PCR (RT-PCR). ( a ) Expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the podocytes. The expression levels differed significantly between the high glucose-induced and wortmannin-treated diabetic groups of podocytes. ( b ) The expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the kidneys of diabetic rats. In addition, wortmannin ameliorated the expression of all podocyte-specific proteins including GLCCI1. ( c , d ) The relative band intensity of proteins in podocytes and the kidneys of diabetic rats was observed by RT-PCR. The band intensity was measured using the Multi Gauge V3.0 software (Fuji Film). *** P

    Journal: Experimental & Molecular Medicine

    Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    doi: 10.1038/emm.2016.28

    Figure Lengend Snippet: Detection of glucocorticoid-induced transcript 1 (GLCCI1) and podocyte-specific marker gene expression by reverse transcription-PCR (RT-PCR). ( a ) Expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the podocytes. The expression levels differed significantly between the high glucose-induced and wortmannin-treated diabetic groups of podocytes. ( b ) The expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the kidneys of diabetic rats. In addition, wortmannin ameliorated the expression of all podocyte-specific proteins including GLCCI1. ( c , d ) The relative band intensity of proteins in podocytes and the kidneys of diabetic rats was observed by RT-PCR. The band intensity was measured using the Multi Gauge V3.0 software (Fuji Film). *** P

    Article Snippet: Podocytes from the three groups were incubated in culture medium containing either 5 or 25 mM glucose for 24 h. After 24 h of incubation, the wortmannin group incubated with high glucose was treated with wortmannin (0.32 μM , LC Laboratories, Woburn, MA, USA); the half-maximal inhibitory concentration of wortmannin was determined by CCK-8 assay (data not shown).

    Techniques: Marker, Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Software

    Localization of glucocorticoid-induced transcript 1 (GLCCI1) in podocyte foot processes (FPs) by immunoelectron microscopy. ( a ) FPs formed by normal podocytes in the control group. ( b ) Effacement of FPs by podocyte injury in the diabetic group. Podocytes were disrupted by streptozotocin (STZ) treatment. ( c ) Podocyte FPs were regulated around the glomerular basement membrane (GBM) by treatment with wortmannin. Wortmannin could restore podocytes to their original condition. ( d ) Immunogold particles (arrowheads) marking GLCCI1 (10 nm gold) were observed in podocyte FPs of the control group. ( e ) No labeling was observed in podocyte FPs of the diabetic group. ( f ) Localization of GLCCI1 in podocyte FPs was observed in the wortmannin-treated diabetic group. Scale bar, 200 nm.

    Journal: Experimental & Molecular Medicine

    Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

    doi: 10.1038/emm.2016.28

    Figure Lengend Snippet: Localization of glucocorticoid-induced transcript 1 (GLCCI1) in podocyte foot processes (FPs) by immunoelectron microscopy. ( a ) FPs formed by normal podocytes in the control group. ( b ) Effacement of FPs by podocyte injury in the diabetic group. Podocytes were disrupted by streptozotocin (STZ) treatment. ( c ) Podocyte FPs were regulated around the glomerular basement membrane (GBM) by treatment with wortmannin. Wortmannin could restore podocytes to their original condition. ( d ) Immunogold particles (arrowheads) marking GLCCI1 (10 nm gold) were observed in podocyte FPs of the control group. ( e ) No labeling was observed in podocyte FPs of the diabetic group. ( f ) Localization of GLCCI1 in podocyte FPs was observed in the wortmannin-treated diabetic group. Scale bar, 200 nm.

    Article Snippet: Podocytes from the three groups were incubated in culture medium containing either 5 or 25 mM glucose for 24 h. After 24 h of incubation, the wortmannin group incubated with high glucose was treated with wortmannin (0.32 μM , LC Laboratories, Woburn, MA, USA); the half-maximal inhibitory concentration of wortmannin was determined by CCK-8 assay (data not shown).

    Techniques: Immuno-Electron Microscopy, Labeling

    Activation of PI3K/Akt and MEK/ERK signaling pathways by direct interaction of PSaV in the absence of GCDCA. (A and B) LLC-PK cells were incubated with PSaV (MOI of 1 FFU/cell) in the absence of GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C and D) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C (C) or transfected with or without siRNAs against PI3K p85α or MEK (D) and then infected with or without PSaV in the absence of GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (E and F) LLC-PK cells were incubated with PSaV VLPs (10 μg/ml), and the cell lysates were harvested at 5 mpi and prepared for Western blotting as described above. (G and H) LLC-PK cells were mock pretreated or pretreated with wortmannin or U0126 at the indicated doses for 1 h at 37°C (G) or transfected with or without siRNAs against PI3K p85α or MEK (H) and then incubated with or without PSaV VLPs. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Activation of PI3K/Akt and MEK/ERK signaling pathways by direct interaction of PSaV in the absence of GCDCA. (A and B) LLC-PK cells were incubated with PSaV (MOI of 1 FFU/cell) in the absence of GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C and D) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C (C) or transfected with or without siRNAs against PI3K p85α or MEK (D) and then infected with or without PSaV in the absence of GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (E and F) LLC-PK cells were incubated with PSaV VLPs (10 μg/ml), and the cell lysates were harvested at 5 mpi and prepared for Western blotting as described above. (G and H) LLC-PK cells were mock pretreated or pretreated with wortmannin or U0126 at the indicated doses for 1 h at 37°C (G) or transfected with or without siRNAs against PI3K p85α or MEK (H) and then incubated with or without PSaV VLPs. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Incubation, Western Blot, Transfection, Infection, Expressing

    Inhibition of the PI3K/Akt and MEK/ERK signaling pathways affects PSaV infectivity and viral protein expression. (A to D) LLC-PK cells were pretreated with noncytotoxic concentrations of wortmannin or U0126 for 1 h at 37°C and then infected with PSaV (MOI of 1 FFU/cell) for 36 h in the presence (A and B) or absence (C and D) of 200 μM GCDCA. (A and C) Levels of PSaV VPg protein were determined by Western blotting. GAPDH was used as a loading control. The intensity of VPg relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane. (B and D) Viral titers were determined by TCID 50 assay. The data are presented as means and standard deviations of the results of three independent experiments. Differences were evaluated using one-way analysis of variance. *, P

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Inhibition of the PI3K/Akt and MEK/ERK signaling pathways affects PSaV infectivity and viral protein expression. (A to D) LLC-PK cells were pretreated with noncytotoxic concentrations of wortmannin or U0126 for 1 h at 37°C and then infected with PSaV (MOI of 1 FFU/cell) for 36 h in the presence (A and B) or absence (C and D) of 200 μM GCDCA. (A and C) Levels of PSaV VPg protein were determined by Western blotting. GAPDH was used as a loading control. The intensity of VPg relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane. (B and D) Viral titers were determined by TCID 50 assay. The data are presented as means and standard deviations of the results of three independent experiments. Differences were evaluated using one-way analysis of variance. *, P

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Inhibition, Infection, Expressing, Western Blot

    PSaV-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were inoculated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then infected with or without PSaV in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 min postinoculation (mpi). The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells transfected with scrambled siRNA (Scram) or siRNA against PI3K p85α or MEK were harvested at 24 and 48 h posttransfection. The downregulation of each protein by siRNA knockdown was evaluated by Western blotting using antibodies specific for each protein. GAPDH was used as a loading control. (E) LLC-PK cells transfected with or without each siRNA were incubated with PSaV (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were determined by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: PSaV-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were inoculated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then infected with or without PSaV in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 min postinoculation (mpi). The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells transfected with scrambled siRNA (Scram) or siRNA against PI3K p85α or MEK were harvested at 24 and 48 h posttransfection. The downregulation of each protein by siRNA knockdown was evaluated by Western blotting using antibodies specific for each protein. GAPDH was used as a loading control. (E) LLC-PK cells transfected with or without each siRNA were incubated with PSaV (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were determined by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Western Blot, Infection, Expressing, Transfection, Incubation

    Inhibition of PSaV trafficking by blockade of PI3K/Akt and MEK/ERK signaling pathways. (A) LLC-PK cells were incubated with Alexa Fluor 594 (AF594)-labeled PSaV particles (approximately 415 particles per cell) for the indicated times at 37°C in the presence of 200 μM GCDCA. The cells were then fixed, permeabilized, and further incubated with a monoclonal antibody against the early endosome marker EEA1 or the late endosome marker LAMP2. After incubation with a FITC-conjugated anti-mouse IgG antibody, the cells were processed for confocal microscopy to determine the colocalization of AF594-labeled PSaV particles with the early endosome marker EEA1 or the late endosome marker LAMP2. The boxed areas are magnified and shown under each panel. (B and C) LLC-PK cells were pretreated with or without wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) for 1 h at 37°C and then infected with AF594-labeled PSaV particles (approximately 415 particles per cell) in the presence (B) or absence (C) of 200 μM GCDCA for 3 h. After fixation and permeabilization, the cells were incubated with a monoclonal antibody against EEA1 or LAMP2 and then with a FITC-conjugated secondary antibody to visualize colocalization of AF594-labeled PSaV particles with EEA1 or LAMP2. The boxed areas are magnified and shown under each panel. All experiments were performed in triplicate, and a representative set of results is shown. Bars, 10 μm (A) and 20 μm (B and C). (D and E) Quantification of AF595-labeled PSaV particles colocalized with the early endosome marker EEA1 (D) and the late endosome marker LAMP2 (E) was performed using 10 confocal microscopy images of cells treated under the conditions described above by use of the ImageJ program. Quantification of signals was made with a threshold of 0.03 to 1.3 µm 2 as described in Materials and Methods.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Inhibition of PSaV trafficking by blockade of PI3K/Akt and MEK/ERK signaling pathways. (A) LLC-PK cells were incubated with Alexa Fluor 594 (AF594)-labeled PSaV particles (approximately 415 particles per cell) for the indicated times at 37°C in the presence of 200 μM GCDCA. The cells were then fixed, permeabilized, and further incubated with a monoclonal antibody against the early endosome marker EEA1 or the late endosome marker LAMP2. After incubation with a FITC-conjugated anti-mouse IgG antibody, the cells were processed for confocal microscopy to determine the colocalization of AF594-labeled PSaV particles with the early endosome marker EEA1 or the late endosome marker LAMP2. The boxed areas are magnified and shown under each panel. (B and C) LLC-PK cells were pretreated with or without wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) for 1 h at 37°C and then infected with AF594-labeled PSaV particles (approximately 415 particles per cell) in the presence (B) or absence (C) of 200 μM GCDCA for 3 h. After fixation and permeabilization, the cells were incubated with a monoclonal antibody against EEA1 or LAMP2 and then with a FITC-conjugated secondary antibody to visualize colocalization of AF594-labeled PSaV particles with EEA1 or LAMP2. The boxed areas are magnified and shown under each panel. All experiments were performed in triplicate, and a representative set of results is shown. Bars, 10 μm (A) and 20 μm (B and C). (D and E) Quantification of AF595-labeled PSaV particles colocalized with the early endosome marker EEA1 (D) and the late endosome marker LAMP2 (E) was performed using 10 confocal microscopy images of cells treated under the conditions described above by use of the ImageJ program. Quantification of signals was made with a threshold of 0.03 to 1.3 µm 2 as described in Materials and Methods.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Inhibition, Incubation, Labeling, Marker, Confocal Microscopy, Infection

    Bile acid-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were treated with or without 200 μM GCDCA and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were determined by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells were transfected with or without siRNAs against PI3K p85α or MEK and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Journal: Journal of Virology

    Article Title: Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells

    doi: 10.1128/JVI.01674-18

    Figure Lengend Snippet: Bile acid-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were treated with or without 200 μM GCDCA and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were determined by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells were transfected with or without siRNAs against PI3K p85α or MEK and then treated with or without 200 μM GCDCA. Cell lysates were harvested at 5 min posttreatment. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.

    Article Snippet: Wortmannin (PI3K inhibitor) and U0126 (MEK inhibitor) were purchased from Invivogen (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).

    Techniques: Activation Assay, Western Blot, Expressing, Transfection

    Local treatment with protein degradation inhibitors leads to dendritic MAPT mislocalization and spine loss. Rat hippocampal neurons (DIV 21–25) cultured in microfluidic chambers were treated on the neuritic side for 24 h either with DMSO (control, a), with the autophagy inhibitor wortmannin (b) or with the proteasomal inhibitor epoxomicin (c). MAP2 antibody (green), K9JA antibody (red) and DBN1 (cyan) were used to label dendrites, MAPT and spines, respectively. Only magnified images of the neuritic side are shown here. (a) In control, neurons treated with vehicle at the neuritic side (DMSO,

    Journal: Autophagy

    Article Title: Pathological missorting of endogenous MAPT/Tau in neurons caused by failure of protein degradation systems

    doi: 10.1080/15548627.2018.1509607

    Figure Lengend Snippet: Local treatment with protein degradation inhibitors leads to dendritic MAPT mislocalization and spine loss. Rat hippocampal neurons (DIV 21–25) cultured in microfluidic chambers were treated on the neuritic side for 24 h either with DMSO (control, a), with the autophagy inhibitor wortmannin (b) or with the proteasomal inhibitor epoxomicin (c). MAP2 antibody (green), K9JA antibody (red) and DBN1 (cyan) were used to label dendrites, MAPT and spines, respectively. Only magnified images of the neuritic side are shown here. (a) In control, neurons treated with vehicle at the neuritic side (DMSO,

    Article Snippet: All protein degradation inhibitors including wortmannin (Merck Millipore, 681,675; 1 µM), bafilomycin A1 (Merck Millipore, 196,000; 0.2 µM), epoxomicin (Merck Millipore, 324,800; 0.2 µM) and lactacystin (Merck Millipore, 426,100; 0.5 µM), protein synthesis inhibitors including cycloheximide (Sigma Aldrich, C4859; 10 µM) and anisomycin (Sigma Aldrich, A5862; 10 µM), modulators of degradation such as trehalose (Sigma Aldrich, 6138–23-4; 150 mM) and rolipram (Enzo Life Sciences, BML-PD175-0010; 10 µM) were added only to the neuritic side and thus had no direct effects on cell bodies.

    Techniques: Cell Culture

    Protein translation inhibitors prevent MAPT missorting. Rat hippocampal neurons (DIV 21–25) cultured in MFCs treated on the neuritic side with the protein translation inhibitors cycloheximide (CHX) or anisomycin (Ani) combined either with wortmannin (a and c) or with epoxomicin (b and d) for 24 h or with Mapt ] = 0.58; p = 0.68). (f–h) When the neurons were treated with siRNA against Mapt in the neuritic side (1 µM, 72 h) (f) there was very low accumulation of MAPT in dendrites on the neuritic side (see merged images in the right panels [g] and quantification [h]). Scale bars in all overview images on left: 20 µm; in all magnifications of insets on right: 5 µm. Error bar, SEM from n = > 800 dendrites/dendritic branches from 4 experiments.

    Journal: Autophagy

    Article Title: Pathological missorting of endogenous MAPT/Tau in neurons caused by failure of protein degradation systems

    doi: 10.1080/15548627.2018.1509607

    Figure Lengend Snippet: Protein translation inhibitors prevent MAPT missorting. Rat hippocampal neurons (DIV 21–25) cultured in MFCs treated on the neuritic side with the protein translation inhibitors cycloheximide (CHX) or anisomycin (Ani) combined either with wortmannin (a and c) or with epoxomicin (b and d) for 24 h or with Mapt ] = 0.58; p = 0.68). (f–h) When the neurons were treated with siRNA against Mapt in the neuritic side (1 µM, 72 h) (f) there was very low accumulation of MAPT in dendrites on the neuritic side (see merged images in the right panels [g] and quantification [h]). Scale bars in all overview images on left: 20 µm; in all magnifications of insets on right: 5 µm. Error bar, SEM from n = > 800 dendrites/dendritic branches from 4 experiments.

    Article Snippet: All protein degradation inhibitors including wortmannin (Merck Millipore, 681,675; 1 µM), bafilomycin A1 (Merck Millipore, 196,000; 0.2 µM), epoxomicin (Merck Millipore, 324,800; 0.2 µM) and lactacystin (Merck Millipore, 426,100; 0.5 µM), protein synthesis inhibitors including cycloheximide (Sigma Aldrich, C4859; 10 µM) and anisomycin (Sigma Aldrich, A5862; 10 µM), modulators of degradation such as trehalose (Sigma Aldrich, 6138–23-4; 150 mM) and rolipram (Enzo Life Sciences, BML-PD175-0010; 10 µM) were added only to the neuritic side and thus had no direct effects on cell bodies.

    Techniques: Cell Culture

    The 200-17-induced FAS and FASL phosphorylation was dependent on PI3K/AKT pathway activation in the LC cell lines. (a) AKT, phosphorylation of AKT, phosphorylated FOXO3, FAS, and FASL, as well as the gray intensity analysis in Hep-2 cells, stimulated with 200-17 for 0.5 h, 1 h, 4 h, 6 h, and 8 h, following pretreatment with wortmannin for 0.5 h. (b) AKT, phosphorylation of AKT, phosphorylated FOXO3, FAS, and FASL, as well as the gray intensity analysis in Tu212 cells, stimulated with 200-17 for 0.5 h, 1 h, 4 h, 6 h, and 8 h, following pretreatment with wortmannin for 0.5 h. n = 3 per group. One representative experiment of the three independent experiments is demonstrated. ∗ P

    Journal: Journal of Immunology Research

    Article Title: IL-17 Affects the Progression, Metastasis, and Recurrence of Laryngeal Cancer via the Inhibition of Apoptosis through Activation of the PI3K/AKT/FAS/FASL Pathways

    doi: 10.1155/2020/2953191

    Figure Lengend Snippet: The 200-17-induced FAS and FASL phosphorylation was dependent on PI3K/AKT pathway activation in the LC cell lines. (a) AKT, phosphorylation of AKT, phosphorylated FOXO3, FAS, and FASL, as well as the gray intensity analysis in Hep-2 cells, stimulated with 200-17 for 0.5 h, 1 h, 4 h, 6 h, and 8 h, following pretreatment with wortmannin for 0.5 h. (b) AKT, phosphorylation of AKT, phosphorylated FOXO3, FAS, and FASL, as well as the gray intensity analysis in Tu212 cells, stimulated with 200-17 for 0.5 h, 1 h, 4 h, 6 h, and 8 h, following pretreatment with wortmannin for 0.5 h. n = 3 per group. One representative experiment of the three independent experiments is demonstrated. ∗ P

    Article Snippet: The PI3K inhibitor, wortmannin, was purchased from the Beyotime Institute of Biotechnology (Shanghai, China).

    Techniques: Activation Assay

    Effects of wortmannin on TGF-β2-induced Smad-mut/Luc promoter activity in ARPE-19 cells. Transfected cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. Results are expressed relative to untreated control cells. Values represent the means ± SD of three independent experiments. * P

    Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

    Article Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-?2 in human retinal pigment epithelial cells

    doi: 10.1007/s00417-011-1766-x

    Figure Lengend Snippet: Effects of wortmannin on TGF-β2-induced Smad-mut/Luc promoter activity in ARPE-19 cells. Transfected cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. Results are expressed relative to untreated control cells. Values represent the means ± SD of three independent experiments. * P

    Article Snippet: The medium was changed to serum-free DMEM/F12 for 24 h, followed by incubation in the presence or absence of 10 ng/ml of TGF-β2, with or without wortmannin, in medium containing 200 μM of L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Activity Assay, Transfection, Incubation

    Effects of wortmannin on TGF-β2-induced CAGA12-Luc activity in ARPE-19 cells. Transfected cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/m L ) for 24 hours. Results are expressed relative to untreated control cells. Values represent the means ± SD of three independent experiments. * P

    Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

    Article Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-?2 in human retinal pigment epithelial cells

    doi: 10.1007/s00417-011-1766-x

    Figure Lengend Snippet: Effects of wortmannin on TGF-β2-induced CAGA12-Luc activity in ARPE-19 cells. Transfected cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/m L ) for 24 hours. Results are expressed relative to untreated control cells. Values represent the means ± SD of three independent experiments. * P

    Article Snippet: The medium was changed to serum-free DMEM/F12 for 24 h, followed by incubation in the presence or absence of 10 ng/ml of TGF-β2, with or without wortmannin, in medium containing 200 μM of L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Activity Assay, Transfection, Incubation

    Effects of wortmannin on TGF-β2-induced Smad7 mRNA expression. Quiescent cell were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. The relative levels of mRNA were normalized against GAPDH from the same cDNA preparation. Values represent the means ± SD of three independent experiments. * P

    Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

    Article Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-?2 in human retinal pigment epithelial cells

    doi: 10.1007/s00417-011-1766-x

    Figure Lengend Snippet: Effects of wortmannin on TGF-β2-induced Smad7 mRNA expression. Quiescent cell were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. The relative levels of mRNA were normalized against GAPDH from the same cDNA preparation. Values represent the means ± SD of three independent experiments. * P

    Article Snippet: The medium was changed to serum-free DMEM/F12 for 24 h, followed by incubation in the presence or absence of 10 ng/ml of TGF-β2, with or without wortmannin, in medium containing 200 μM of L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Expressing, Incubation

    PI3K p85 and Akt activation induced by TGF-β2 in ARPE-19 cells. a PI3K p85 activation: lysates from cells pretreated with or without wortmannin (10 μM) and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 5 min or 24 h. Bands corresponding to the phosphorylated form of PI3K p85 ( top ) and total PI3K p85 ( bottom ) were detected. Wortmannin did not inhibit TGF-β2-induced PI3K p85 activation. b Akt activation: lysates from cells incubated in the presence of TGF-β2 with or without pretreatment with wortmannin were analyzed using a kit. Wortmannin inhibited TGF-β2-induced Akt activation. Data represent the means ± SD of three independent experiments. * P

    Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

    Article Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-?2 in human retinal pigment epithelial cells

    doi: 10.1007/s00417-011-1766-x

    Figure Lengend Snippet: PI3K p85 and Akt activation induced by TGF-β2 in ARPE-19 cells. a PI3K p85 activation: lysates from cells pretreated with or without wortmannin (10 μM) and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 5 min or 24 h. Bands corresponding to the phosphorylated form of PI3K p85 ( top ) and total PI3K p85 ( bottom ) were detected. Wortmannin did not inhibit TGF-β2-induced PI3K p85 activation. b Akt activation: lysates from cells incubated in the presence of TGF-β2 with or without pretreatment with wortmannin were analyzed using a kit. Wortmannin inhibited TGF-β2-induced Akt activation. Data represent the means ± SD of three independent experiments. * P

    Article Snippet: The medium was changed to serum-free DMEM/F12 for 24 h, followed by incubation in the presence or absence of 10 ng/ml of TGF-β2, with or without wortmannin, in medium containing 200 μM of L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Activation Assay, Incubation

    Effects of wortmannin on TGF-β2-induced COL1A1 and COL1A2 mRNA expression. Quiescent cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. The relative levels of mRNA were normalized against GAPDH from the same cDNA preparation. Values represent the means ± SD of three independent experiments. * P

    Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

    Article Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-?2 in human retinal pigment epithelial cells

    doi: 10.1007/s00417-011-1766-x

    Figure Lengend Snippet: Effects of wortmannin on TGF-β2-induced COL1A1 and COL1A2 mRNA expression. Quiescent cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. The relative levels of mRNA were normalized against GAPDH from the same cDNA preparation. Values represent the means ± SD of three independent experiments. * P

    Article Snippet: The medium was changed to serum-free DMEM/F12 for 24 h, followed by incubation in the presence or absence of 10 ng/ml of TGF-β2, with or without wortmannin, in medium containing 200 μM of L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Expressing, Incubation

    Wortmannin reduces the production of TGF-β2-induced type I collagen in ARPE-19 cells. Cells were preincubated with vehicle or wortmannin ( c ) for 30 min and incubated in the absence ( a ) or presence ( b , c ) of TGF-β2 (10 ng/mL) for 24 h. Bar :100 μm

    Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

    Article Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-?2 in human retinal pigment epithelial cells

    doi: 10.1007/s00417-011-1766-x

    Figure Lengend Snippet: Wortmannin reduces the production of TGF-β2-induced type I collagen in ARPE-19 cells. Cells were preincubated with vehicle or wortmannin ( c ) for 30 min and incubated in the absence ( a ) or presence ( b , c ) of TGF-β2 (10 ng/mL) for 24 h. Bar :100 μm

    Article Snippet: The medium was changed to serum-free DMEM/F12 for 24 h, followed by incubation in the presence or absence of 10 ng/ml of TGF-β2, with or without wortmannin, in medium containing 200 μM of L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Incubation

    Effects of wortmannin on TGF-β2-induced COL1A2 promoter activity in ARPE-19 cells. Transfected cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. Results are expressed relative to untreated control cells. Values represent the means ± SD of three independent experiments. * P

    Journal: Graefe's Archive for Clinical and Experimental Ophthalmology

    Article Title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-?2 in human retinal pigment epithelial cells

    doi: 10.1007/s00417-011-1766-x

    Figure Lengend Snippet: Effects of wortmannin on TGF-β2-induced COL1A2 promoter activity in ARPE-19 cells. Transfected cells were pretreated with or without wortmannin and incubated in the presence or absence of TGF-β2 (10 ng/ml) for 24 hours. Results are expressed relative to untreated control cells. Values represent the means ± SD of three independent experiments. * P

    Article Snippet: The medium was changed to serum-free DMEM/F12 for 24 h, followed by incubation in the presence or absence of 10 ng/ml of TGF-β2, with or without wortmannin, in medium containing 200 μM of L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Activity Assay, Transfection, Incubation

    NF-κB activation by EVs relied on a dynamin-dependant endocytosis. (A) NF-κB activation by 1 µg.ml −1 EVs for 24 h in T84 cells pre-treated 2 h before with 20µM dynasore (N = 3). (B) NF-κB activation by 1 µg.ml −1 EV for 24 h in T84 cells pre-treated 2 h before with endocytosis inhibitors wortmannin 20 µM, genistein 50 µM, chlorpromazine 5 µM, pitstop2 20 µM, or Cdc42 inhibitor casin 10 µM (N ≥ 3). Data are expressed as median ± quartiles of fold change toward unstimulated cells.

    Journal: Frontiers in Immunology

    Article Title: Fusobacterium nucleatum Extracellular Vesicles Modulate Gut Epithelial Cell Innate Immunity via FomA and TLR2

    doi: 10.3389/fimmu.2020.583644

    Figure Lengend Snippet: NF-κB activation by EVs relied on a dynamin-dependant endocytosis. (A) NF-κB activation by 1 µg.ml −1 EVs for 24 h in T84 cells pre-treated 2 h before with 20µM dynasore (N = 3). (B) NF-κB activation by 1 µg.ml −1 EV for 24 h in T84 cells pre-treated 2 h before with endocytosis inhibitors wortmannin 20 µM, genistein 50 µM, chlorpromazine 5 µM, pitstop2 20 µM, or Cdc42 inhibitor casin 10 µM (N ≥ 3). Data are expressed as median ± quartiles of fold change toward unstimulated cells.

    Article Snippet: Inhibitors were added 2 h prior testing: 1 µM TAK-242 TLR4 inhibitor (Calbiochem, # 243984-11-4), 20 µM Dynasore (Sigma-Aldrich, # 324410), 1 µM CU-CTP22 TLR1/TLR2 antagonist (Merck Millipore # 614305), Genistein 50 µM (Abcam # ab120112), Wortmannin 20µM (Abcam # ab120148), Chlorpromazine hydrochloride (Sigma # C0982), PitStop2 (Sigma # SML 1169), and Casin (Sigma # SML 1253).

    Techniques: Activation Assay