Journal: Scientific Reports

Article Title: Activation of NADPH oxidases leads to DNA damage in esophageal cells

doi: 10.1038/s41598-017-09620-4

Figure Lengend Snippet: PKCα and PKCβ isoforms are involved in activation of NAD(P)H oxidases and generation of ROS in BA/A-treated cells. ( A ) CP-A cells were pre-treated with the indicated kinase inhibitors for 1 hour, treated with BA/A (100 μM, pH 4.0) and analyzed for phosphorylation of p47 phox subunit in the cellular membrane fraction. Only PKC inhibitor Go6976 efficiently blocked phosphorylation of p47 phox in BA/A-treated cells. ( B ) Acidic bile salts induce phosphorylation of PKC isoforms. CP-A cells were treated with BA/A and analyzed for PKCα(Ser657) and PKCβ(Thr641) phosphorylation at the indicated time. ( C ) Acidic bile salts induce translocation of PKCα and PKCβ to the cellular membrane. CP-A cells were treated with BA/A and analyzed for the subcellular localization of PKCα and PKCβ. As a positive control, CP-A cells were treated with PMA. ( D ) Inhibition of PKC isoforms decreases levels of ROS after treatment with BA/A. ROS levels were measured by flow cytometry using DCFDA staining in CP-A cells treated with BA/A. Top panels: measurement of ROS levels after inhibition of PKCα and PKCβ with Go6976 and Go6983 compounds. Representative flow cytometry profiles are shown. Bottom panels: measurement of ROS levels after downregulation of PKCα and PKCβ with the specific siRNA. Representative flow cytometry profiles are shown. Downregulation of PKCα and PKCβ is shown on the right bottom panel. Bar graphs show mean values ± S.E. of three independent experiments (*p < 0.05, n = 3). ROS levels in control cells were arbitrarily set to 1. ( E ) Downregulation of PKCα and PKCβ with siRNAs suppresses phosphorylation of p47 phox in BA/A-treated cells. CP-A cells, in which PKC isoforms were downregulated, were treated with BA/A for 30 min and analyzed for p47 phox phosphorylation in the cellular membrane fraction. Na/K-ATPase was used as a loading control. Right panel shows levels of PKCα and PKCβ after downregulation with siRNA.

Article Snippet: The following chemicals were used: NAC (N-acetylcysteine, Sigma-Aldrich), apocynin (4-hydroxy-3-methoxyacetophenone, Sigma-Aldrich), tempol (4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl, Enzo Biochem, Farmingdale, NY), MitoTEMPO ((2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride, Enzo Biochem), Go6976 (5,6,7,13-Tetrahydro-13-methyl-5-oxo-12 H -indolo[2,3- a ]pyrrolo[3,4- c ]carbazole-12-propanenitrile, PKC inhibitor, TOCRIS Bioscience, Bristol, UK), LY 294002 (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, PI3K inhibitor, Calbiochem, San Diego, CA), Erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine, EGFR inhibitor, Santa Cruz Biotechnology), MK-2206 2HCl (Akt inhibitor, Selleckchem), Go6983 (3-[1-[3-(Dimethylamino)propyl]-5-methoxy-1 H -indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione, PKC inhibitor, Selleckchem), PMA (12-O-tetradecanoylphorbol 13-acetate, Sigma-Aldrich).

Techniques: Activation Assay, Phospho-proteomics, Membrane, Translocation Assay, Positive Control, Inhibition, Flow Cytometry, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy

doi: 10.1074/jbc.m111.269134

Figure Lengend Snippet: FIGURE 1. Gö6976, Jak3 inhibitor VI and KU55933 inhibit autophagosome formation. A, MCF7, HeLa, and U-2-OS cells infected with RLuc-LC3wt or RLuc-LC3G120A expressing retroviruses were left untreated (Control) or starved for 4 h (HBSS) in the presence or absence () of 8 M Gö6976, 2 M KU55933 or 5 M Jak3 inhibitor VI. The data represent average S.D. of at least three independent experiments. B, quantification and a representative immunoblot with antibodies against p62 and GAPDH (loading control) in total extracts from MCF7 cells cultured in normal growth medium (control), starved for 7 h in the absence (HBSS) or presence of 10 mM 3-MA, 2 M KU55933, 8 M Gö6976, or 5 M Jak3 inhibitor VI. The data represent the p62-signal normalized to GAPDH. The untreated control was defined as 100%. The data represent average S.D. of four independent experiments. C, average number of autophagosomes/cell S.E. in MCF7-eGFP-LC3 cells left untreated or treated as indicated with 200 nM rapamycin, 2 M KU55933, 10 M Gö6976, or 5 M Jak3 inhibitor VI for 6 h. D, immunoblotting with antibodies against LC3 and GAPDH in total extracts from MCF7 cells cultured in normal growth medium (control) or treated for 3h with rapamycin (100 nM) concanamycin A (2 nM) in the absence or presence of 10 mM 3-MA, 2 M KU55933, 8 M Gö6976, or 5 M Jak3 inhibitor VI. Quantification of the LC3-II signal normalized to GAPDH signal is shown below. *, p value 0.05; **, p value 0.01; and ***, p value 0.001 as compared with control (A) or control (B, HBSS) or HBSS (B, HBSSinhibitors) or rapamycin (C) in the absence of the inhibitors.

Article Snippet: Reagents and Treatments—The following reagents were used: Small molecule kinase inhibitor library-I and II (Calbiochem, Cat. 539744 and 539745), KU55933 (Tocris Biosciences, Cat. 3544), Gö6976 (Tocris Biosciences, Cat. 2253), CP466722 (Axon Medchem, axon 1495), LY294002 (Sigma, L9908), Jak3 Inhibitor VI (Calbiochem, 420126), Rapamycin (Sigma R0395), 3-MA (Sigma, M9281), zVAD-fmk (Bachem, Switzerland), concanamycin A (Sigma) Necrostatin-1 (Sigma N9037).

Techniques: Infection, Expressing, Control, Western Blot, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy

doi: 10.1074/jbc.m111.269134

Figure Lengend Snippet: FIGURE 3. KU55933 and Gö6976 inhibit Vps34. A, confocal microscopy images of GFP-2XFYVE expressing MCF7 cells either untreated (Control) or treated for 2 h with 2 M KU55933, 8 M Gö6976, 5 M Jak3 inhibitor VI, 10 mM 3-MA, or 10 mM caffeine. The images were converted to grayscale and inverted using Photoshop. B, comparison of the effects of KU55933 (left) and Gö6976 (right) on rapamycin-induced autophagy and on eGFP-2XFYVE-relocalization. (B, top panels) MCF7 cells expressing RLucLC3wt or RLuc-LC3G120A were left untreated or treated with 100 nM rapamycin alone or in combination with indicated concentrations of KU55933 or Gö6976 for 6 h. The data represent average S.D. of at least three independent experiments. (B, bottom panels) MCF7- eGFP-2XFYVE cells were treated with indicated concentrations of KU55933 or Gö6976 for 2 h and the number of eGFP-positive dots/cell were calculated in confocal images. *, p value 0.05; **, p value 0.01; and ***, p value 0.001 as compared with cells treated with rapamycin alone (B, top) or control (B, bottom). C, immunoblotting with antibodies against Vps34 and Beclin 1 of streptactin-pulldowns of MCF7 cells transiently transfected with pEXPR-IBA105 (Str.), pStrep-VPS34wt (Str.WT), pStrep-VPS34D743N (Str. D743N), or pStrep-VPS34L750M (Str. L750M). The bottom row shows immunoblotting of the corresponding lysates with antibody against Beclin 1. D, dot blotting of PtdIns(3)P produced in vitro by strep-tagged Vps34WT (Str.WT) or strep-tagged Vps34L750M (Str.L750M). Kinase reactions were performed in the absence of inhibitors (Control) or with either KU55933, LY-294002, or Gö6976 at the indicated concentra- tions. The bar graph shows the Quantification of PtdIns(3)P dot-blots from Vps34 in vitro kinase reactions analyzing either Strep-tagged Vps34wt or Strep- tagged Vps34L750M. Kinase reactions were performed in the absence of inhibitors (Control) or with KU55933 (n 3), LY-294002 (Vps34wt) (n 3) LY-294002 (Vps34L750M) (n 2), or Gö6976 (n 2) as indicated. E, immunoblotting with antibodies against Akt and phospho-Akt in total extracts from MCF7 cells incubated with the indicated concentrations of inhibitors for 6 h.

Article Snippet: Reagents and Treatments—The following reagents were used: Small molecule kinase inhibitor library-I and II (Calbiochem, Cat. 539744 and 539745), KU55933 (Tocris Biosciences, Cat. 3544), Gö6976 (Tocris Biosciences, Cat. 2253), CP466722 (Axon Medchem, axon 1495), LY294002 (Sigma, L9908), Jak3 Inhibitor VI (Calbiochem, 420126), Rapamycin (Sigma R0395), 3-MA (Sigma, M9281), zVAD-fmk (Bachem, Switzerland), concanamycin A (Sigma) Necrostatin-1 (Sigma N9037).

Techniques: Confocal Microscopy, Expressing, Control, Comparison, Western Blot, Transfection, Produced, In Vitro, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Ginsenoside-Rd Promotes Neurite Outgrowth of PC12 Cells through MAPK/ERK- and PI3K/AKT-Dependent Pathways

doi: 10.3390/ijms17020177

Figure Lengend Snippet: Effects of Rd on ERK, AKT and PKC phosphorylation. Western blot showed the changes in the phosphorylation levels of ERK1/2 (p-ERK1/2, a ), AKT (p-AKT, b ), PKC (p-PKC, c ) in PC12 cells. Total protein levels of ERK1/2, AKT, and PKC were used as respective internal controls. PD, PD98059; LY, LY294002; Go, Go6976. * p < 0.05 ( vs. the control group); # p < 0.01 ( vs. the Rd group).

Article Snippet: PI3K/AKT kinase inhibitor LY294002, MAPK/ERK1/2 kinase inhibitor PD98059, and PKC kinase inhibitor Go6976 were from Promega (Madison, WI, USA), and prepared in the saline containing 0.1% DMSO (Sigma-Aldrich).

Techniques: Phospho-proteomics, Western Blot, Control