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: Cell Death Discovery

Article Title: Macrophage-derived ectosomal miR-350-3p promotes osteoarthritis progression through downregulating chondrocyte H3K36 methyltransferase NSD1

doi: 10.1038/s41420-024-01986-5

Figure Lengend Snippet: A KEGG pathways enrichment analysis of the differentially genes in chondrocytes overexpressed by miR-350-3p mimics. B The binding site between miR-350-3p and NSD1 predicted by ENCORI database. C – E , H Quantitative PCR and western blot of NSD1, H3K36me1, H3K36me2 in chondrocytes treated with miR-350-3p mimics or mimics-NC (CON) and miR-350-3p inhibitor or inhibitor-NC (CON). F , G IHC staining and quantification of NSD1 in cartilage. I Immunoblotting of NSD1, COL2, MMP13, P16 and P21 in chondrocytes treated with si-NSD1 or si-NC. J , K Immunoblotting of NSD1, COL2, MMP13, P16, P21, H3K36me1 and H3K36me2 in chondrocytes treated with Tensile-Ectos, si-NSD1 and miR-350-3p inhibitor. Statistical analyses were conducted using Student’s t test ( F ), two-way analysis of variance followed by Sidak’s multiple comparison test ( C , H ), one-way analysis of variance followed by Tukey’s multiple comparison test ( G ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant.

Article Snippet: Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16 INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Comparison

Journal: Cell Death Discovery

Article Title: Macrophage-derived ectosomal miR-350-3p promotes osteoarthritis progression through downregulating chondrocyte H3K36 methyltransferase NSD1

doi: 10.1038/s41420-024-01986-5

Figure Lengend Snippet: MiR-350-3p increases in macrophages induced by excessive mechanical loading and is delivered to chondrocytes via ectosomes. Elevated miR-350-3p in chondrocytes promotes the catabolism and aging of chondrocytes by inhibiting the expression of NSD1 and down-regulating the methylation level of H3K36, and accelerates the progression of OA.

Article Snippet: Subsequently, the protein levels were measured by Fdbio-Dura ECL (Fdbio-Science) after incubation with specific secondary antibodies in the membrane for 1 h. The antibodies used in this study for western blot include: HSP70 (1:1000, Abcam, ab181606), CD81 (1:1000, Ptoteintech, 66866-1), Calnexin (1:500, Ptoteintech, 10427-2), COL2 (1:1000, Abclonal, A1560), Tsg101 (1:1000, Abcam, ab125011), MMP13 (1:1000, Abclonal, A11148), p16 INK4a (1:2000, Abcam, ab211542), NSD1 (1:500, Abclonal, A9981), Mono Methyl-Histone H3-K36 (1:2000, Abclonal, A22863), DiMethyl-Histone H3-K36 (1:10000, Abclonal, A22087), Histone H3 (1:2000, Abclonal, A17562), HRP-labeled goat IgG H&L (Jackson ImmunoResearch), GAPDH (1:8000, Proteintech, 60004-1-Ig), and P21 (1:1000, Abcam, ab109199).

Techniques: Expressing, Methylation