Journal: Mediators of Inflammation

Article Title: Anti-Inflammatory Mechanism of Polyunsaturated Fatty Acids in Helicobacter pylori -Infected Gastric Epithelial Cells

doi: 10.1155/2014/128919

Figure Lengend Snippet: Effects of PUFAs on PKC δ activation in H. pylori -stimulated AGS cells. (a) AGS cells were infected with H. pylori for the indicated time points (4, 8, 15, 30, and 60 min). Phosphospecific and total forms of PKC δ were determined by western blotting. (b) AGS cells were pretreated with various concentrations of rottlerin (0.04, 0.2, and 1 μ M), a specific PKC δ inhibitor, for 1 h and then cultured with H. pylori for 30 min. Phosphospecific and total forms of ERK and JNK in whole cell extracts were determined by western blotting. (c) AGS cells were pretreated with individual fatty acid (100 μ M) for 24 h and were then cultured with H. pylori for 8 min.

Article Snippet: The proteins were detected with antibodies against ERK, JNK, or phospho-JNK (Cell Signaling Technology, Beverly, MA, USA) and phospho-ERK, PKC δ , phospho-PKC δ , EGFR, and phospho-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted in TBS-T containing 3–5% dry milk, and incubated at 4°C for 16 h. After washing with TBS-T, primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (anti-goat or anti-rabbit) and visualized using the ECL detection system (Santa Cruz Biotechnology), according to the manufacturer's instruction.

Techniques: Activation Assay, Infection, Western Blot, Cell Culture

Journal: Mediators of Inflammation

Article Title: Anti-Inflammatory Mechanism of Polyunsaturated Fatty Acids in Helicobacter pylori -Infected Gastric Epithelial Cells

doi: 10.1155/2014/128919

Figure Lengend Snippet: Effect of a PKC δ inhibitor on EGFR activation and effects of inhibitors of EGFR, PKC δ , ERK, and JNK on H. pylori -stimulated IL-8 production. (a) AGS cells were preincubated with various doses of rottlerin (0.04, 0.2, and 1 μ M) for 1 h and were then cultured with H. pylori for 15 min. Phosphospecific and total forms of PKC δ were determined by western blotting. (b) AGS cells were preincubated with AG1478 (1 μ M), rottlerin (1 μ M), U0126 (20 μ M), and SP600125 (20 μ M) for 1 h and were then infected with H. pylori for 4 h. IL-8 levels in the medium were measured by enzyme-linked immunosorbent assay. Each bar represents mean ± S.E. of four separate experiments. * P < 0.01 versus None; + P < 0.01 versus control.

Article Snippet: The proteins were detected with antibodies against ERK, JNK, or phospho-JNK (Cell Signaling Technology, Beverly, MA, USA) and phospho-ERK, PKC δ , phospho-PKC δ , EGFR, and phospho-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted in TBS-T containing 3–5% dry milk, and incubated at 4°C for 16 h. After washing with TBS-T, primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (anti-goat or anti-rabbit) and visualized using the ECL detection system (Santa Cruz Biotechnology), according to the manufacturer's instruction.

Techniques: Activation Assay, Cell Culture, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: TLR4 counteracts BVRA signaling in human leukocytes via differential regulation of AMPK, mTORC1 and mTORC2

doi: 10.1038/s41598-019-43347-8

Figure Lengend Snippet: Our working model for biliverdin/BVRA and TLR4 signaling in leukocytes. Biliverdin induced responses are shown in black. Biliverdin acts on BVRA. Our data suggest that biliverdin facilitates formation of BVRA signaling complex(s) that include mTORC2. mTORC2 induces phosphorylation of two targets: PKCζ at Thr410 and Akt at Ser473. PKCζ is involved in LKB1 Ser431 and AMPKα Thr172 phosphorylation. AMPK phosphorylates TSC1/2 at Ser1387 and Raptor at Ser792. Therefore, BVRA activation may contribute to mTORC1 inhibition via two SI: phosphorylated Raptor, which inhibits mTORC1 complex formation, and TSC1/2 mediated inhibition of mTORC1 activity. Because mTORC1 is central to TLR4 signaling in leukocytes , BVRA inhibition of mTORC1 suppresses TLR4. Pharmacological inhibitors are shown in blue. Torin and PP242, but not rapamycin, inhibited BVRA-mediated protein-protein interactions. TLR4 induced response are shown in red. Once activated, TLR4 down-regulates BVRA and PKCζ expression (dashed lines). TLR4 upregulates expression of MMP9 , which then triggers AMPKα cleavage (dashed line), leading to Raptor Ser792 dephosphorylation . This enables mTORC1 activation, which then triggers an increase in HIF-1α expression and S6K1 phosphorylation at Thr389. TLR4 also triggers an increase in haptoglobin expression, thus limiting heme availability. Using these parallel mechanisms TLR4 ensures biliverdin/BVRA and mTORC2 signaling inhibition, and on the other hand, mTORC1 activation.

Article Snippet: HIF-1α (sc-10790; 1:250), AMPKα (sc-25792; 1:1000), MMP9 (sc-10737; 1:1000), TLR4 (sc-10741; 1:500), BVRA (sc-393385; 1:1000), Akt1 (sc-5928; 1:500), p-LKB1 Ser431 (sc-271924; 1:500), LKB1 (sc-32245; 1:500), p-CaMKKβ Thr286 (sc-32289; 1:750), CaMKKβ (sc-100364; 1:1000), p-mTOR Ser2481 (sc-293132; 1:1000), mTOR (sc-517464; 1:1000), p-PKCζ Thr410 (sc-271962; 1:500), PKCζ (sc-393218; 1:500), heme oxygenase 1 (HO-1) (sc-136960; 1:1000), heme oxygenase 2 (HO-2) (sc-17786; 1:1000), tuberin (TSC1/2)(sc-271314; 1:500), haptoglobin β (sc-390962; 1:500), p-NOS3 Ser1177 (sc-81510; 1:500) and NOS3 (sc-376751; 1:500) were all from Santa Cruz Biotechnology. p-Raptor Ser792 (#2083; 1:1000), Raptor (#2280; 1:200), p-p70 S6 kinase Thr389 (#9205; 1:1000), p-AMPKα Thr172 (#2535; 1:1000), p-Akt Ser473 (#4051; 1:1000), p-Akt Thr308 (#9275; 1:1000), tuberin/TSC2 (#4308; 1:1000) and p-tuberin/TSC2 Ser1387 (#5584; 1:1000) were all from Cell Signaling Technology.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Activity Assay, Protein-Protein interactions, Expressing, De-Phosphorylation Assay

Journal: Oncology reports

Article Title: Dual inhibition of FOXM1 and its compensatory signaling pathway decreased the survival of ovarian cancer cells.

doi: 10.3892/or.2020.7845

Figure Lengend Snippet: Figure 4. Proteins expression in ovarian cancer cells treated with inhibitors and FDI‑6. (A) Western blot analysis of N‑Ras expression in HeyA8 cells treated with tipifarnib and FDI‑6 in three‑dimensional cell culture. (B) Western blot analysis of proteins that are associated with the HER3 downstream signaling pathway in HeyA8 cells treated with sapitinib. (C) ATP levels in HeyA8 cells treated with rottlerin. Rottlerin inhibits the activity of the kinase that transfers a phosphate group to PKCδ. Error bars represent standard deviation. All experiments were performed in duplicate. (D) Western blot analysis of N‑Ras expression in CAOV3 cells treated with tipifarnib. FDI‑6, forkhead domain inhibitor‑6; p, phosphorylated.

Article Snippet: Membranes were incubated with primary antibodies against FOXM1 (cat. no. 5436; Cell Signaling Technology, Inc.), HER3 (cat. no. 12708; Cell Signaling Technology, Inc.), N-Ras (cat. no. sc-31; Santa Cruz Biotechnology, Inc.), p-PKCδ (S664) (cat. no. sc-365463; Santa Cruz Biotechnology, Inc.), PKCδ (cat. no. ab18216; Abcam), and β-actin (cat. no. A5441; Sigma-Aldrich; Merck KGaA) were diluted 1:1,000 in TBS with 0.1% Tween‐20 (TBS‐T) overnight at 4 ̊C and then washed three times with TBS-T.

Techniques: Expressing, Western Blot, Cell Culture, Activity Assay, Standard Deviation

Journal: Oncology reports

Article Title: Dual inhibition of FOXM1 and its compensatory signaling pathway decreased the survival of ovarian cancer cells.

doi: 10.3892/or.2020.7845

Figure Lengend Snippet: Figure 3. Protein expression in ovarian cancer cells treated with FDI‑6. Western blot analysis of N‑Ras, p‑PKCδ (S664), PKCδ and HER3 proteins in (A) HeyA8 and (B) CAOV3 cells treated with 1, 3 or 10 µM FDI‑6 in three‑dimensional cell culture for 24 h. FDI‑6, forkhead domain inhibitor‑6.

Article Snippet: Membranes were incubated with primary antibodies against FOXM1 (cat. no. 5436; Cell Signaling Technology, Inc.), HER3 (cat. no. 12708; Cell Signaling Technology, Inc.), N-Ras (cat. no. sc-31; Santa Cruz Biotechnology, Inc.), p-PKCδ (S664) (cat. no. sc-365463; Santa Cruz Biotechnology, Inc.), PKCδ (cat. no. ab18216; Abcam), and β-actin (cat. no. A5441; Sigma-Aldrich; Merck KGaA) were diluted 1:1,000 in TBS with 0.1% Tween‐20 (TBS‐T) overnight at 4 ̊C and then washed three times with TBS-T.

Techniques: Expressing, Western Blot, Cell Culture

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Penh responses to aerosolized methacholine in control mice (square) and OVA-sensitized/challenged mice (circle) treated with vehicle (black) or H89 (10 mg/kg) (grey) in the acute ( A ) and moderate ( B ) asthma models. (B = baseline). Data represent mean values ± SEM (bars) from n = 6−9 mice for control groups and n = 12 mice for OVA sensitized/challenged groups (OVA). * P <0.05 and *** P <0.001 vs corresponding controls; ### P <0.001 vs group indicated; NS: not significant.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Effect of H89 (10 mg/kg) (grey blocks) or vehicle (black blocks) on total leukocyte (Total), eosinophil (Eos) and macrophage (Mac) numbers in BAL fluid 24 hours after the last challenge in control (Ctr) and OVA-sensitized/challenged mice (OVA) in the acute ( A ) and moderate ( B ) asthma models. C–D. Effect of H89 (10 mg/kg) (grey blocks) or vehicle (black blocks) on neutrophils (Neu) and lymphocytes (Lym) numbers in BAL fluid 24 hours after the last challenge in control (Ctr) and OVA-sensitized/challenged mice (OVA) in the acute ( C ) and moderate ( D ) asthma models. Data represent mean values (blocks) ± SEM (bars) from n = 6−9 mice per group (saline) and n = 9−12 mice per group (OVA). * P <0.05, ** P <0.01 and *** P <0.001 vs corresponding controls; ## P <0.01 and ### P <0.001 vs group indicated; NS: not significant.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Control, Saline

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: H&E-stained lung sections demonstrating peribronchial inflammatory infiltrates 24 hours after the last OVA challenge (magnification × 200). ( B–C ) Inflammation score in lung sections from control (Ctr) and OVA sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks) in the acute ( B ) and moderate ( C ) asthma models. Data represent mean values ± SEM (bars) from n = 6 mice per group. *** P <0.001 vs corresponding controls; ### P <0.001 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Periodic acid Schiff (PAS)-stained lung sections demonstrating hyperplasia of mucus-producing goblet cells 24 hours after the last OVA challenge (magnification × 200). ( B–C ) Mucus score in lung sections from control (Ctr) and OVA sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks) in the acute ( B ) and moderate ( C ) asthma models. Data represent mean values ± SEM (bars) from n = 6 mice per group. ** P <0.01 and *** P <0.001 vs corresponding controls; ## P <0.01 and ### P <0.001 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Acidic toluidine blue-stained lung sections 24 hours after the last OVA challenge (magnification × 200). Black arrows indicate toluidine blue-positive mast cells ( B–C ) Quantification of mast cell numbers in lung sections from control (Ctr) and OVA sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks) in the acute ( B ) and moderate ( C ) asthma models. Data represent mean values ± SEM (bars) from n = 6 mice per group. ** P <0.01 and *** P <0.001 vs corresponding controls; ## P <0.01 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: BAL fluid was collected 24 hours after the last OVA challenge. The levels of IL-4 ( A–B ) and IL-5 ( C–D ) were determined using ELISA in the acute ( A & C ) and moderate ( B & D ) asthma models in control (Ctr) and OVA-sensitized/challenged (OVA) mice treated with vehicle (black blocks) or H89 (grey blocks). Data represent mean values ± SEM (bars) from n = 6−8 mice per group. * P <0.05, ** P <0.01 and *** P <0.001 vs corresponding controls; # P <0.05 and ## P <0.01 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

doi: 10.1371/journal.pone.0049512

Figure Lengend Snippet: Serum was collected 24 hours after the last OVA challenge. The levels of OVA-specific IgE ( A–B ), OVA-specific IgG1 ( C–D ), OVA-specific IgG2a ( E ) and OVA-specific IgG2c ( F ) were determined using ELISA in the acute ( A, C & E ) and moderate ( B, D & F ) asthma models in control (squares) and OVA sensitized/challenged (circles) mice treated with vehicle (black blocks) or H89 (grey blocks). Data represent mean values ± SEM (bars) from n = 4−6 mice per group (saline) and n = 6−12 mice per group (OVA). * P <0.05, ** P <0.01 and *** P <0.001 vs corresponding controls; # P <0.05 and ## P <0.01 vs group indicated.

Article Snippet: H89 ( N -[2-( p -Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt) (10 mg/kg) (LC Laboratories, PKC Pharmaceuticals Inc., Woburn, MA, USA) suspended in 5% DMSO in saline was administered i.p. two hours before each OVA challenge (or two hours before the last OVA challenge only for ).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Saline