Journal: Chemical Science

Article Title: Universal cell membrane camouflaged nano-prodrugs with right-side-out orientation adapting for positive pathological vascular remodeling in atherosclerosis †

doi: 10.1039/d4sc00761a

Figure Lengend Snippet: Synthesis and characterization of OEM-ETBNPs. (a) Schematic diagram of right-side-out-orientated coupling of ETBNPs onto the inner leaflet of the OEM. (b) WB analysis of protein expression after lentivirus transfection. (c) Quantification analysis of (b). (d) Flow cytometry of anti-integrin α9 on the EM and OEM. (e) Quantification analysis of (d). (f) The mass ratio of OEM-ETBNPs and EM-ETBNPs. (g) Hydrodynamic diameter of ETBNPs, EM-ETBNPs, and OEM-ETBNPs. (h) Zeta potentials of ETBNPs, EM-ETBNPs, and OEM-ETBNPs. (i) Representative TEM images of ETBNPs, cell membrane–ETBPD, and cell membrane-ETBNPs. (j) Representative confocal images of the DiO-cell membrane and Cy5-PS-targeted peptide. (k) Flow cytometry analysis of the DiO-cell membrane and Cy5-PS-targeted peptide modified ETBNPs. Data shown as mean ± SD. Statistical analysis: (c) one-way ANOVA with Tukey's post hoc test; n = 3. (e) Student's t -test; n = 3; *** P < 0.0001.

Article Snippet: The Annexin V-Cy5 apoptosis detection kit, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD) dyes, and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) dyes were obtained from Biotium Inc (Fremont, USA).

Techniques: Expressing, Transfection, Flow Cytometry, Membrane, Modification

Journal: Chemical Science

Article Title: Universal cell membrane camouflaged nano-prodrugs with right-side-out orientation adapting for positive pathological vascular remodeling in atherosclerosis †

doi: 10.1039/d4sc00761a

Figure Lengend Snippet: Characterization of the right-side-out orientation of the cell membrane. (a) Schematic diagram of the PS distribution of OEM-ETBNPs. (b) Representative confocal images of PS distribution in OEM-ETBNPs using the probe Annexin V-Cy5. (c) Flow cytometry of PS distribution in OEM-ETBNPs. (d) Quantification analysis of (c). (e) Schematic illustration of the cell membranes with ssDNA probes and an ssDNA quencher for investigating the right-side-out orientation of OEM-ETBNPs. (f) Quantifying the different right-side-out orientations of probe-ETBNPs and probe-ETBPD with FRET. Data shown as mean ± SD. Statistical analysis: (d) one-way ANOVA with Tukey's post hoc test; n = 5. (f) Student's t -test; n = 8. *** P < 0.0001.

Article Snippet: The Annexin V-Cy5 apoptosis detection kit, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD) dyes, and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) dyes were obtained from Biotium Inc (Fremont, USA).

Techniques: Membrane, Flow Cytometry

Journal: Chemical Science

Article Title: Universal cell membrane camouflaged nano-prodrugs with right-side-out orientation adapting for positive pathological vascular remodeling in atherosclerosis †

doi: 10.1039/d4sc00761a

Figure Lengend Snippet: Characterization of the target drug delivery and therapeutic efficacy of AS in ApoE −/− mice. (a) Blood circulation measurements of free Cy5, Cy5-labeled EM-ETBNPs, and OEM-ETBNPs in vivo. (b) Ex vivo images of whole blood collected after tail vein administration of free Cy5, Cy5-labeled EM-ETBNPs, and OEM-ETBNPs. (c) Ex vivo images of aorta treated with free Cy5, Cy5-labeled EM-ETBNPs, and OEM-ETBNPs at 12 h and 24 h. (d) Schematic of the animal experimental procedure. (e) Representative photographs of en face ORO-stained aortas and quantitative analysis of the lipid content in (j). (f) ORO-stained cross-sections of aortic roots. (g) H&E-stained cross-sections of aortic roots. (h) Toluidine blue-stained cross-sections of aortic roots. (i) Masson-stained cross-sections of aortic roots. (k) Quantification analysis of lipid content in (f). (l) Quantification analysis of inflammation in (g). (m) Quantification analysis of the necrotic nucleus in (h). (n) Quantification analysis of collage content in (i). Data shown as mean ± SD. Statistical analysis: (j–n) one-way ANOVA with Tukey's post hoc test; n = 5 and *** P < 0.0001. For (j), * P = 0.0346 and * P = 0.0269. For (k), ** P = 0.0099. For (l), ** P = 0.0075 and * P = 0.0264. For (n), ** P = 0.0046.

Article Snippet: The Annexin V-Cy5 apoptosis detection kit, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD) dyes, and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) dyes were obtained from Biotium Inc (Fremont, USA).

Techniques: Labeling, In Vivo, Ex Vivo, Staining

Journal: Frontiers in Chemistry

Article Title: Surface Functionalization of Organosilica Nanoparticles With Au Nanoparticles Inhibits Cell Proliferation and Induces Cell Death in 4T1 Mouse Mammary Tumor Cells for DNA and Mitochondrial-Synergized Damage in Radiotherapy

doi: 10.3389/fchem.2022.907642

Figure Lengend Snippet: (A) FM images of 4T1 cells stained blue with DAPI (indicates the cell nucleus) and stained magenta with Cy5 (indicates the γ-H2AX) treated with 30 μg ml −1 of NP (-), OS, OS/PEI, and OS/Au/PEI with X-ray irradiation after 1 h. Scale bar = 50 μm. (B) γ-H2AX expression in 4T1 cells treated with 30 μg ml −1 of NP (-), OS, OS/PEI, and OS/Au/PEI with X-ray irradiation after 1 and 6 h * Significantly different from the 4T1 cells treated with 0 Gy NP (-). # Significantly different from the 4T1 cells treated with 8 Gy NP (-). † Significantly different from the 4T1 cells treated with 8 Gy OS/PEI.

Article Snippet: Annexin V-Cy5/DAPI assay: At the desired time, the cells were stained using the Annexin V-Cy5 apoptosis detection kit (Bio-Vision, Inc. Milpitas, CA, United States), followed by DAPI staining.

Techniques: Staining, Irradiation, Expressing

Journal: Frontiers in Chemistry

Article Title: Surface Functionalization of Organosilica Nanoparticles With Au Nanoparticles Inhibits Cell Proliferation and Induces Cell Death in 4T1 Mouse Mammary Tumor Cells for DNA and Mitochondrial-Synergized Damage in Radiotherapy

doi: 10.3389/fchem.2022.907642

Figure Lengend Snippet: (A) FM image of 4T1 cells for apoptotic and necrotic cell determination by Annexin V/DAPI staining treated with 30 μg ml −1 of NP (-), OS, OS/PEI, and OS/Au/PEI with X-ray irradiation after DAY4. Scale bar = 50 μm. (B) The FM image analysis of 4T1 cells for apoptotic and necrotic cell determination by Annexin V/DAPI staining treated with 30 μg ml −1 of NP (-), OS, OS/PEI, and OS/Au/PEI with X-ray irradiation after DAY1 and DAY4. * Significantly different from the 4T1 cells treated with 0 Gy NP (-). # Significantly different from the 4T1 cells treated with 8 Gy NP (-). † Significantly different from the 4T1 cells treated with 8 Gy OS/PEI.

Article Snippet: Annexin V-Cy5/DAPI assay: At the desired time, the cells were stained using the Annexin V-Cy5 apoptosis detection kit (Bio-Vision, Inc. Milpitas, CA, United States), followed by DAPI staining.

Techniques: Staining, Irradiation

Journal: PLOS One

Article Title: Rosuvastatin protects against oxLDL-induced endothelial cell oxidative stress and attenuates atherosclerotic plaque formation in ApoE -/- mice through the NF-κB pathway

doi: 10.1371/journal.pone.0339967

Figure Lengend Snippet: (A-B) Bcl2 and Bax mRNA expression in HUVECs treated with different concentrations of rosuvastatin and ox-LDL (200 µg/ml) for 24 h. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA. (C-D) BCL-2 and Bax protein expression in HUVECs treated with different concentrations of rosuvastatin and ox-LDL (200 µg/ml) for 24 h. The data are presented as the means ± SEMs. * P < 0.05 by one-way ANOVA. (E) HUVECs stimulated with ox-LDL and different concentrations of rosuvastatin were stained with DAPI (blue), Bax (green) and mitochondria (red); scale bar = 20 μm. (F) Early and late apoptosis of HUVECs treated with 100 µg/mL ox-LDL and 10 μmol/L rosuvastatin for 24 h. The quantification results are shown on the right (n = 5). *** P < 0.001, **** P < 0.0001 by one-way ANOVA.

Article Snippet: The next day, the cell supernatant and adherent cells were collected and stained in the dark at 4°C for 15 minutes using an Annexin V-APC/PI Apoptosis Kit (FITC and PerCP-Cy5.5, Elabscience, E-CK-A217).

Techniques: Expressing, Staining

Journal: PLOS One

Article Title: Rosuvastatin protects against oxLDL-induced endothelial cell oxidative stress and attenuates atherosclerotic plaque formation in ApoE -/- mice through the NF-κB pathway

doi: 10.1371/journal.pone.0339967

Figure Lengend Snippet: (A-D) Protein levels of IkBα, p-IkBα, P65 and p-P65 in HUVECs treated with or without 10 µM rosuvastatin and treated with 100 µg/mL ox-LDL for 24 h. The data are presented as the means ± SEMs. * P < 0.05, ** P < 0.01 by one-way ANOVA. (E) Schematic diagram illustrating the role of rosuvastatin in ox-LDL-induced endothelial cell dysfunction. Rosuvastatin regulates oxidative stress and apoptosis-related gene transcription in endothelial cells by inhibiting ox-LDL-induced IKBα and P65 activation in endothelial cells.

Article Snippet: The next day, the cell supernatant and adherent cells were collected and stained in the dark at 4°C for 15 minutes using an Annexin V-APC/PI Apoptosis Kit (FITC and PerCP-Cy5.5, Elabscience, E-CK-A217).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

doi: 10.1038/s41467-026-72524-3

Figure Lengend Snippet: a Residues along the LRRC58-CDO1 interface of the structure representing CDO1 (orange) ubiquitylation by LRRC58 (purple) with neddylated CUL5-RBX2-ARIH2 are labeled and shown in full opacity. Residues found mutated in patients are labeled in bold. b The LRRC58-CDO1 interface is subdivided into patches. D-patch residues, involved in degrader-induced interaction with VHL, are shown with respect to the interface patches on the right. CDO1 patch schematic shown below. c CDO1-Cmpd8-VHL-EloB/C crystal structure (PDB:8VL9, CDO1 light gray, VHL dark gray, Compound 8 (Cmpd8) white) aligned to CDO1 (orange) of the sample representing CDO1 ubiquitylation by LRRC58 with neddylated CUL5-RBX2-ARIH2. Hydrogen bonding interactions are shown (black dotted lines). d Cmpd8-mediated recruitment of CDO1 to VHL orients CDO1 differently than LRRC58, shown by alignment of a chimeric structure of VHL-Cmpd8-CDO1 (PDB:8VL9) in complex with CUL2 NTD -EloB/C (PDB:4WQO) to the fitted model of the LRRC58-CDO1-CUL2 complex. On left, lysines are colored as dark-blue spheres; Lys8 position is highlighted in a dark-blue circle. Sites of patient mutations, H147 and E143 (green and pink spheres, respectively), are highlighted with black circles, on right. e Summary of CDO1 mutations used. f Reporter stability for CDO1 variants expressed in HEK293T cells grown in complete or cysteine-free media. All mutations made to CDO1 patches lose sensitivity to cysteine starvation, whereas D-patch mutations respond. g Reporter stability as in ( d ) except comparing cells gown in complete media and treated with Cmpd8 or DMSO. All CDO1 variants are efficiently degraded, including the patient variants, except for D-patch mutations. h In vitro reconstituted assays comparing WT and mutant Cy5-CDO1 ubiquitylation by neddylated LRRC58-CUL2. Mutations only in patch interfaces, including patient variants, reduced CDO1 ubiquitylation. i Same as in ( h ) except with neddylated VHL-CUL2 and Cmpd8. CDO1 patch mutants, and all patient variants, show WT-like ubiquitylation efficiencies, but D-patch mutants display reduced ubiquitylation. In ( f ) and ( g ), the average values from n = 4 independent replicates are shown with error bars reporting standard deviation. Fluorescence scans in ( h ) and ( i ) are representative of n = 2 technical replicates. Source data provided as Source Data file.

Article Snippet: For the assays comparing ubiquitylation of CDO1 variants by the VHL-Cmpd8 degrader, components were mixed as previously described, but with either Cy5-CDO1 or Cy5-K8R-CDO1 (0.25 μM), either LRRC58-EloB/C or VHL-EloB/C (0.5 μM), either WT-ubiquitin or K 0 -ubiquitin (100 μM), and either DMSO or Cmpd8 (0.125 μM, NVS-VHL720-MedChemExpress).

Techniques: Labeling, In Vitro, Mutagenesis, Standard Deviation, Fluorescence

Journal: PLoS Pathogens

Article Title: Differential Gene Expression Patterns of EBV Infected EBNA-3A Positive and Negative Human B Lymphocytes

doi: 10.1371/journal.ppat.1000506

Figure Lengend Snippet: (A) EBNA-3A negative LCLs proliferate at reduced rates. Three independent wt and EBNA-3A negative LCLs derived from two individual donors were seeded at an initial density of 2×10 5 cells per ml and viable cell counts were determined over a period of three weeks. Results are given as total numbers of viable cells corrected for the expansion of the cultures over time. The data are shown as mean values of triplicates. (B) EBNA-3A negative LCLs show reduced S-phase entry compared to wt LCLs. The cell cycle status of wt and EBNA-3A negative LCLs derived from two different donors was determined with the thymidine analogue BrdU, which was added to the respective cultures for 2 hrs prior to FACS analysis. The incorporated BrdU was stained with an APC-coupled anti-BrdU antibody and total DNA was counterstained with 7-AAD. Cells were inspected for G 0 /G 1 , S and G 2 /M phases of the cell cycle and for sub G1 DNA content by FACS analysis. (C) EBNA-3A negative LCLs exhibit higher levels of apoptotic cells compared to wt LCLs. The fraction of apoptotic cells in cultures of wt and EBNA-3A negative LCLs was determined by FACS analysis after staining of cells with Cy5-coupled Annexin V and 7-AAD. Cells that stain positive for Annexin V-Cy5 but negative for 7-AAD are in early apoptosis, while cells that stain positive for both are either in the end stage of apoptosis or dead.

Article Snippet: BrdU-assays (APC BrdU Flow kit; BD Biosciences) and Annexin V-apoptosis assays (Annexin V-Cy5 and 7-AAD; BD Biosciences) were performed according to the manufacturer's protocol.

Techniques: Derivative Assay, Staining