Journal: Theranostics

Article Title: A MAM-targeting therapeutic peptide restores autophagy homeostasis and ameliorates atherosclerosis

doi: 10.7150/thno.132357

Figure Lengend Snippet: Peptide 4 activates the AMPK-TFEB axis and induces functional autophagy. Human umbilical vein endothelial cells (HUVECs) were treated with Peptide 4 (100 µM) or non-treated (NT) for the indicated durations prior to analysis. (A) Measurement of intracellular ATP levels following Peptide 4 treatment to assess cellular energy status. (B) Immunoblot analysis of AMPK activation, shown as phosphorylation of AMPK (p-AMPK) and total AMPK levels. (C) Immunoblot analysis of AMPK activation in cells treated with Peptide 4 in the presence of Compound C (Com. C; 20 µM) or STO-609 (10 µM). (D) Immunocytochemical analysis of TFEB subcellular localization following Peptide 4 treatment. Nuclei were counterstained with DAPI (blue). Representative images and quantification of TFEB nuclear translocation are shown. Scale bar, 20 µm. (E) Lysosomal content assessed by LysoTracker Red (LTR) staining in control (NT) and Peptide 4-treated cells (12 h). Representative images and quantification are shown. Scale bar, 20 µm. (F) Tandem fluorescent LC3 reporter analysis of autophagic flux following Peptide 4 treatment (12 h). Representative images and quantification of LC3 puncta are shown. Autophagosomes were defined as GFP⁺/mRFP⁺ puncta, and autolysosomes as mRFP-only puncta. Scale bar, 10 µm. (G) Immunoblot analysis of LC3B-I/LC3B-II conversion and SQSTM1/p62 levels following Peptide 4 treatment (100 µM) over the indicated time points (24-72 h). (H) Immunoblot analysis of AMPK activation following siRNA-mediated knockdown of GRP75 in HUVECs. (I) Immunocytochemical analysis of TFEB localization following GRP75 knockdown in HUVECs. Nuclei were counterstained with DAPI (blue). Representative images and quantification of TFEB nuclear translocation are shown. Scale bar, 20 µm. (J) Immunoblot analysis of LC3B-I/LC3B-II conversion and SQSTM1/p62 levels following GRP75 knockdown in HUVECs. Data are presented as mean ± SEM. Statistical significance was defined as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Article Snippet: For pharmacological modulation experiments, cells were pretreated with inhibitors such as chloroquine (CQ; C6628, Sigma-Aldrich, St. Louis, MO), Compound C (P5499, Sigma-Aldrich), or STO-609 (HY-19805, MedChemExpress, Princeton, NJ) for the indicated durations, prior to or together with peptide treatment, as indicated in the corresponding figure legends.

Techniques: Functional Assay, Western Blot, Activation Assay, Phospho-proteomics, Translocation Assay, Staining, Control, Knockdown

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: The Journal of Biological Chemistry

Article Title: Ca 2+ /Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth *

doi: 10.1074/jbc.M109.006296

Figure Lengend Snippet: Inhibitors of CaMKs and CaMKK suppress ionomycin-induced cofilin phosphorylation and neurite outgrowth. A, effects of inhibitors on ionomycin-induced cofilin phosphorylation. Neuro-2a cells were pretreated with control DMSO, 3 μm KN-93, or 5 μm STO-609 for 1 h and then stimulated with 1.5 μm ionomycin for 30 min. Cell lysates were analyzed by immunoblotting (IB) with anti-P-cofilin and anti-cofilin antibodies. The relative P-cofilin levels are shown as means ± S.D. of triplicate experiments. **, p < 0.001. B, effects of inhibitors on ionomycin-induced neurite outgrowth. Neuro-2a cells transfected with CFP were pretreated with control DMSO, KN-93, or STO-609 and stimulated with ionomycin as in A. Cells were cultured for 48 h and fixed, and their CFP fluorescence was visualized. The arrows indicate neurite-bearing cells. Scale bar, 10 μm. C, quantitative analysis of the data shown in B. The number of neurite-bearing cells was analyzed as in Fig. 3A. Data represent means ± S.D. of triplicate experiments (60–80 cells in each experiment). *, p < 0.01.

Article Snippet: KN-93 and STO-609 were purchased from Wako (Osaka, Japan).

Techniques: Western Blot, Transfection, Cell Culture, Fluorescence