Journal: Journal of Biological Chemistry

Article Title: Human T Cell Activation Results in Extracellular Signal-regulated Kinase (ERK)-Calcineurin-dependent Exposure of Tn Antigen on the Cell Surface and Binding of the Macrophage Galactose-type Lectin (MGL)*

doi: 10.1074/jbc.m113.471045

Figure Lengend Snippet: FIGURE 6. MGL ligand expression is controlled by an ERK- and calcineurin-dependent pathway. A–E, CD4 T cells were left untreated or stimulated with CD3/CD28 beads in the presence or absence of PP2 (Src kinases, 12.5 M), GO6983 (PKC, 10 M), FK506 (calcineurin, 10 nM), Bay11-7082 (IB-, 1 M), U0126 (MEK/ERK, 25 M), SB203580 (P38 MAPK, 12.5 M), and SP600125 (JNK, 25 M). DMSO was added as a vehicle control. All inhibitors were titrated and used at concentrations that allowed for at least 90% cell survival. 7-AAD was included to exclude dead cells. Mean fluorescent intensities and percentage of positive cells are indicated in the plots. All inhibitors were tested in at least three independent donors, yielding similar results. One representative donor is shown. FSC, forward scatter.

Article Snippet: The following inhibitor concentrations were used: PP2 (Src kinases, 12.5 M), GO6983 (PKC, 10 M), SR11302 (AP-1, 10 M), FK506 (calcineurin, 10 nM, all from Tocris Biosciences, Bristol, UK), U0126 (MEK/ERK, 25 M), SB203580 (P38 MAPK, 12.5 M), SP600125 (JNK, 25 M) and Bay11-7082 (I B- , 1 M, all from Invivogen, San Diego).

Techniques: Expressing, 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