Journal: PLOS ONE

Article Title: Aβ 25-35 -induced autophagy and apoptosis are prevented by the CRMP2-derived peptide ST2-104 (R9-CBD3) via a CaMKKβ/AMPK/mTOR signaling hub

doi: 10.1371/journal.pone.0309794

Figure Lengend Snippet: SH-SHY5Y cells were treated with Aβ 25–35 or control medium or with ST2-104 peptide for 24 h at 37°C and then protein levels were assessed by Western blotting. Detection of CaMKKβ, AMPK, pAMPK, mTOR and p-mTOR protein expression levels using Western blot analysis. Representative blots are shown. Levels of β-actin protein were used as the loading control. Bar represents mean ± SEM from 3 separate wells. One-way ANOVA with Tukey’s post-hoc tests with * P <0.05, ** P <0.01, vs. control group; # P <0.05, ## P <0.01, vs. Aβ 25–35 group (n = 3 per condition).

Article Snippet: Cells were then challenged with 5 μM Aβ 25–35 (Sigma-Aldrich) and 5 to 40 μM ST2-104 peptide (added as a pre-treatment 30 min (Yaoqiang Biological Company, PR China)) and left for 24 h. In separate experiments, cells were pretreated with the mTOR inhibitor (500 nM Rapamycin; Selleck Chemicals) for 4h or the CaMKKβ inhibitor (5 μM STO-609; Selleck Chemicals) for 1h in RPMI 1640 medium.

Techniques: Control, Western Blot, Expressing

Journal: PLOS ONE

Article Title: Aβ 25-35 -induced autophagy and apoptosis are prevented by the CRMP2-derived peptide ST2-104 (R9-CBD3) via a CaMKKβ/AMPK/mTOR signaling hub

doi: 10.1371/journal.pone.0309794

Figure Lengend Snippet: SH-SHY5Y cells were treated with Aβ 25–35 or control medium or with ST2-104 peptide for 24 h at 37°C and then apoptosis levels and apoptosis-related proteins were assessed. In some wells, 10 μM STO-609, an inhibitor of CaMKKβ was added for 24 h. (A) Apoptosis level was evaluated using the Hoechst 33,258 staining. Scale bar: 20 μm. For each well, at least 5 different fields were examined–a representative is shown here. (B) Detection of Bax, Bcl-2 and C-caspase-3 protein expression levels using Western blot analysis. Representative blots are shown. Levels of β-actin protein were used as the loading control. Bar represents mean ± SEM from 3 separate wells. * P <0.05, ** P <0.01, vs. control group; # P <0.05, ## P <0.01, vs. Aβ 25–35 group.

Article Snippet: Cells were then challenged with 5 μM Aβ 25–35 (Sigma-Aldrich) and 5 to 40 μM ST2-104 peptide (added as a pre-treatment 30 min (Yaoqiang Biological Company, PR China)) and left for 24 h. In separate experiments, cells were pretreated with the mTOR inhibitor (500 nM Rapamycin; Selleck Chemicals) for 4h or the CaMKKβ inhibitor (5 μM STO-609; Selleck Chemicals) for 1h in RPMI 1640 medium.

Techniques: Control, Staining, Expressing, Western Blot

Journal: PLOS ONE

Article Title: Aβ 25-35 -induced autophagy and apoptosis are prevented by the CRMP2-derived peptide ST2-104 (R9-CBD3) via a CaMKKβ/AMPK/mTOR signaling hub

doi: 10.1371/journal.pone.0309794

Figure Lengend Snippet: SH-SHY5Y cells were treated with Aβ 25–35 or control medium or with ST2-104 for 24 h at 37°C and then autophagy levels and autophagy-related proteins were assessed. In some wells, 10 μM STO-609, an inhibitor of CaMKKβ was added for 24 h. (A) Autophagy levels were evaluated using MDC staining. Scale bar: 20 μm. For each well, at least 5 different fields were examined–a representative is shown here. (B) Detection of BEclin-1, LC1 and LC3 protein expression levels using Western blot analysis. Representative blots are shown. Levels of β-actin protein were used as the loading control. Bar represents mean ± SEM from 3 separate wells. * P <0.05, ** P <0.01, vs. control group; # P <0.05, ## P <0.01, vs. Aβ 25–35 group.

Article Snippet: Cells were then challenged with 5 μM Aβ 25–35 (Sigma-Aldrich) and 5 to 40 μM ST2-104 peptide (added as a pre-treatment 30 min (Yaoqiang Biological Company, PR China)) and left for 24 h. In separate experiments, cells were pretreated with the mTOR inhibitor (500 nM Rapamycin; Selleck Chemicals) for 4h or the CaMKKβ inhibitor (5 μM STO-609; Selleck Chemicals) for 1h in RPMI 1640 medium.

Techniques: Control, Staining, Expressing, Western Blot

Journal: PLoS ONE

Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

doi: 10.1371/journal.pone.0170102

Figure Lengend Snippet: Potency of different Kv1.3 blockers on human Kv1.3 channels on transfected CHO cells.

Article Snippet: The Kv1.3 channel blockers used in this report were OsK1 peptide (Alomone Labs), ShK peptide (Bachem), Kv261 peptide (Janssen R&D), and Kv261-HSA-34 fusion protein (Janssen R&D).

Techniques: Transfection

Journal: PLoS ONE

Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

doi: 10.1371/journal.pone.0170102

Figure Lengend Snippet: OsK1 peptide inhibited human Kv1.3 mediated whole cell currents in a concentration dependent manner in human CD4 T cells (A). Potency of OsK1 peptide on Kv1.3 currents in purified human CD4 T cells and Kv1.3 transfected CHO cells were determined and IC 50 curves were shown (B). Whole cell voltage-gated currents were measured in manual patch clamp assays. All recordings were made at room temperature using Multiclamp 700A amplifier and pClamp 9 software as described in Methods and Materials.

Article Snippet: The Kv1.3 channel blockers used in this report were OsK1 peptide (Alomone Labs), ShK peptide (Bachem), Kv261 peptide (Janssen R&D), and Kv261-HSA-34 fusion protein (Janssen R&D).

Techniques: Concentration Assay, Purification, Transfection, Patch Clamp, Software

Journal: PLoS ONE

Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

doi: 10.1371/journal.pone.0170102

Figure Lengend Snippet: Potency and efficacy of Kv1.3 blockers on human T cells under different stimulation conditions.

Article Snippet: The Kv1.3 channel blockers used in this report were OsK1 peptide (Alomone Labs), ShK peptide (Bachem), Kv261 peptide (Janssen R&D), and Kv261-HSA-34 fusion protein (Janssen R&D).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

doi: 10.1371/journal.pone.0170102

Figure Lengend Snippet: Kv1.3 mRNA expression in different human tissues was profiled in Affymetrix microarrays (A). Cell surface Kv1.3 channels were measured with patch express assays on activated or resting human CD4 T cell subsets including naïve, central memory (Tcm) and effector memory (Tem) T cells, as well as antigen-specific T cells from house dust mite (HDM) stimulation. Data shown are either channels/cell (B) or number of channels normalized to cell surface area (channels/pF) (C). Time course of Kv1.3 channel activity and expression of the activation markers CD25 and CD69 are shown on effector memory CD4 T cells activated with anti-CD3/CD28 coated beads (D). Expression of Kv1.3 mRNA levels in naïve, Tcm and Tem CD4 T cells at different time points post activation with anti-CD3/CD28 coated beads are presented (E). Patch express measurement of Kv1.3 channels was performed on T cell subsets purified from 6 healthy blood donors. Statistical significance on the difference in Kv1.3 channel levels between activated and resting T cell subsets was analyzed by 2-tailed Student’s t test and P values were determined and indicated as *P < 0.05, ***P < 0.001, ****P < 0.0001.

Article Snippet: The Kv1.3 channel blockers used in this report were OsK1 peptide (Alomone Labs), ShK peptide (Bachem), Kv261 peptide (Janssen R&D), and Kv261-HSA-34 fusion protein (Janssen R&D).

Techniques: Expressing, Activity Assay, Activation Assay, Purification

Journal: PLoS ONE

Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

doi: 10.1371/journal.pone.0170102

Figure Lengend Snippet: Kv1.3 expression on human primary T cell subsets.

Article Snippet: The Kv1.3 channel blockers used in this report were OsK1 peptide (Alomone Labs), ShK peptide (Bachem), Kv261 peptide (Janssen R&D), and Kv261-HSA-34 fusion protein (Janssen R&D).

Techniques: Expressing

Journal: PLoS ONE

Article Title: T Cell Subset and Stimulation Strength-Dependent Modulation of T Cell Activation by Kv1.3 Blockers

doi: 10.1371/journal.pone.0170102

Figure Lengend Snippet: Kv1.3 blocker OsK1, Kv261 peptides and Kv261-HSA-34 fusion protein blocked proliferation of CD4 T cells from house dust mite allergic patients following 5 day stimulation with house dust mite extracts (A, B). IL-5 production from house dust mite-specific T cells was inhibited by Kv1.3 blockers as shown with OsK1 and Kv261-HSA-34 (C). OsK1 peptide at 0.3μM inhibited partially the proliferation of PBMC from patients allergic to ragweed after 3 day stimulation with ragweed extracts (D). Decreased proliferative response of individual donor PBMC samples by OsK1 peptide was shown in a separated graph. CTLA4-Ig or cyclosporine A (1μM) were used as reference compounds. The assays in panels A-C were repeated in 4 independent experiments with samples in duplicates. Data from one representative experiment were presented. The study in panel D was done in an experiment with 29 subjects per group. Statistical significance on the difference in proliferation between untreated and OsK1 treated groups were analyzed by 2-way ANOVA and the P value was determined and indicated as *P < 0.05. Readouts in (A) and (C) were performed on CD4 T cells from the same donor whereas those in (B) were from a separate donor. Data from one representative experiment were presented.

Article Snippet: The Kv1.3 channel blockers used in this report were OsK1 peptide (Alomone Labs), ShK peptide (Bachem), Kv261 peptide (Janssen R&D), and Kv261-HSA-34 fusion protein (Janssen R&D).

Techniques:

Journal: Crystal Growth & Design

Article Title: Epitaxial Structure of (001)- and (111)-Oriented Perovskite Ferrate Films Grown by Pulsed-Laser Deposition

doi: 10.1021/cg901355c

Figure Lengend Snippet: X-ray pole figures (along the ω- and ϕ-axes) of 222 SrFeO 2.5 reflections for films grown on (111) SrTiO 3 (STO) substrates with (a) small and (b) large spot area. Red and white curves display contour lines leveled at 50% and 10% of peak intensity of the film reflections, respectively. (c and d) Reciprocal space maps around 222 STO reflections taken at ϕ = 0° [incidence parallel to the (11̅0) STO plane] for the films shown in parts a and b, respectively.

Article Snippet: The SFO films were grown on atomically flat (001) and (111) surfaces of STO substrates (Shinkosha, Co., Ltd.) using a PLD system equipped with a semiconductor-laser-diode heater. ( ) KrF excimer laser pulses (248 nm, 4 Hz) were focused on a target (a SFO ceramic tablet, 99.99% purity) with two different spot areas (0.35 × 0.10 cm 2 and 0.70 × 0.17 cm 2 , hereafter referred as to “small” and “large” spot areas, respectively) at a constant fluence of 1 J/cm 2 .

Techniques:

Journal: Crystal Growth & Design

Article Title: Epitaxial Structure of (001)- and (111)-Oriented Perovskite Ferrate Films Grown by Pulsed-Laser Deposition

doi: 10.1021/cg901355c

Figure Lengend Snippet: Reciprocal space maps around 114 SrTiO 3 (STO) reflections for films grown on (001) STO substrates with (a) small and (b) large spot area, and around 330 STO reflections for films grown on (111) STO substrates with (c) small and (d) large spot area. The inset at the right bottom in each panel depicts schematic domain structures. Crossing bars (red) indicate two splitting peaks of the film reflections, and vertical dashed lines (black) are guides for the substrate peak positions. The red horizontal lines shown in parts a and b correspond to the positions of 004 SrFeO 2.5 reflections. The bars shown at the bottom of parts c and d are referred to in the text.

Article Snippet: The SFO films were grown on atomically flat (001) and (111) surfaces of STO substrates (Shinkosha, Co., Ltd.) using a PLD system equipped with a semiconductor-laser-diode heater. ( ) KrF excimer laser pulses (248 nm, 4 Hz) were focused on a target (a SFO ceramic tablet, 99.99% purity) with two different spot areas (0.35 × 0.10 cm 2 and 0.70 × 0.17 cm 2 , hereafter referred as to “small” and “large” spot areas, respectively) at a constant fluence of 1 J/cm 2 .

Techniques: