Journal: The Journal of Cell Biology

Article Title: Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

doi: 10.1083/jcb.200905007

Figure Lengend Snippet: Osh4p has more than one membrane-binding surface. (A) The indicated mutations were introduced into Osh4p lacking endogenous cysteines (cys-less). The proteins were incubated with liposomes with the indicated lipid composition (N-MCC-PE contains a sulfhydryl-reactive headgroup) for 2 h at RT. The amount of protein pelleting with the membranes after washing (P) and the total amount of input protein (T) were determined by SDS-PAGE. The percentage of protein in P is shown ( n = 3). Black lines indicate that intervening lanes have been spliced out. (B) Single-cysteine mutants were allowed to react with a sulfhydryl-reactive NBD derivative. The ratio of the fluorescence of the proteins before (Fo) and after (F) the addition of liposomes (99:1 PS/PI(4,5)P2) was measured ( n = 6–8). (C, left) The structure of Osh4p (blue) bound to cholesterol (yellow) is shown. The flexible lid domain is shown in orange. (middle) A surface rendering of Osh4p, indicating the positively (blue) and negatively (red) charged surface. (right) The opposite side of the protein is shown. The positions of the residues changed to cysteine are indicated, and those that may interact with liposomes are shown in green. Error bars indicate mean ± SEM.

Article Snippet: The NEM-containing phospholipid N-MCC-PE was obtained from Avanti Polar Lipids, Inc. 10 µg Osh4p was incubated with sucrose-loaded liposomes containing PC/PE/MPB-PE/cholesterol/PI(4,5)P2 (59.5:29:1:10:0.5) for 2 h at room temperature.

Techniques: Binding Assay, Incubation, SDS Page, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

doi: 10.1083/jcb.200905007

Figure Lengend Snippet: Cholesterol extraction by Osh4p is regulated by PIPs in a second membrane. (A) 200 pmol Osh4p was incubated at 30°C with 500 µM 99:1 PC/[ 14 C]cholesterol liposomes together with 400 µM auxiliary liposomes containing 100% PC (red squares), 99.5:0.5 PC/PI(4,5)P 2 (blue triangles), or 99:1 PS/PI(4)P (gray triangles). All liposomes were sucrose filled. At the indicated times, the samples were placed on ice, and the liposomes were pelleted. The amount of [ 14 C]cholesterol in the supernatant was determined by scintillation counting. (B) 2 µM Osh4p was incubated at 30°C with 500 µM 99:1 PC/[ 14 C]cholesterol liposomes. After 15 min (arrow), auxiliary liposomes were added, and the amount of [ 14 C]cholesterol extracted was determined as in A ( n = 3). (C) Performed as in B except that auxiliary liposomes were added to a final concentration of 400 µM (1×), 800 µM (2×), or 1.6 mM (4×). The total incubation was 30 min at 30°C ( n = 4). Error bars indicate mean ± SEM.

Article Snippet: The NEM-containing phospholipid N-MCC-PE was obtained from Avanti Polar Lipids, Inc. 10 µg Osh4p was incubated with sucrose-loaded liposomes containing PC/PE/MPB-PE/cholesterol/PI(4,5)P2 (59.5:29:1:10:0.5) for 2 h at room temperature.

Techniques: Incubation, Concentration Assay

Journal: Journal of Inflammation Research

Article Title: Lycopus lucidus Turcz Exerts Neuroprotective Effects Against H 2 O 2 -Induced Neuroinflammation by Inhibiting NLRP3 Inflammasome Activation in Cortical Neurons

doi: 10.2147/JIR.S305031

Figure Lengend Snippet: LLT protects cortical neurons against H 2 O 2 -induced neuronal damage. ( A ) Schematic depiction of experimental design for primary cortical neurons ( B ) Cell viability assay results of cortical neurons treated with various concentrations of LLT without H 2 O 2 induction at 24 h, n = 3. ( C ) CCK-8 results of H 2 O 2 -treated cortical neurons treated with various concentrations of LLT at 24 h, n = 3. ( D ) The total number of live and dead cells calculated from cellular fluorescence images of each group. n = 10. ( E ) The percentage of apoptotic cells analyzed by flow cytometry of Annexin-V-FITC/PI-PE double staining. n = 5. ( F ) Fluorescence images of live and dead (red) cells for analysis of the neuroprotective effect of LLT at different concentrations on H 2 O 2 -treated cortical neurons. Scale bar = 200 µm. ( G ) Representative flow cytometric plot using Annexin-V/PI to confirm the mode of cell death. Blank = non-treatment, H 2 O 2 = H 2 O 2 only, LLT = H 2 O 2 +LLT extract. Significant differences are indicated as follows and were analyzed via one-way ANOVA with Tukey’s post-hoc test: # p < 0.001 vs the blank group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs the H 2 O 2 group.

Article Snippet: Apoptotic cell death was detected using an Annexin V-Fluorescein isothiocyanate (FITC) propidium iodide (PI)-phycoerythrin apoptosis detection kit (Abcam) as described previously.

Techniques: Viability Assay, CCK-8 Assay, Fluorescence, Flow Cytometry, Double Staining

Journal: Translational Oncology

Article Title: Unraveling the anti-cancer potential of procyanidin B2 from grape seeds in gastric cancer through a multi-omics approach with emphasis on ROS and ferroptosis

doi: 10.1016/j.tranon.2025.102642

Figure Lengend Snippet: Procyanidin B2 triggers apoptosis in gastric cancer cells via the mitochondrial pathway. (A) Scheme of PB2 triggers apoptosis in gastric cancer cells. (B) Flow cytometric analysis of cell apoptosis using Annexin V-FITC/PI staining in gastric cancer cells following treatment with different concentrations of PB2 for 24 h. (C) Quantification of apoptosis detected by flow cytometric analysis. (D) Immunofluorescence analysis of Cleaved-Caspase-3 (×400). (E) Quantification of the relative fluorescence intensity of Cleaved-Caspase-3. (F-H) Mitochondrial membrane potential was analyzed by JC-10 staining using flow cytometry (F) and fluorescence microscopy (×400) (G), followed by quantitative analysis (H). (I-J) Immunoblot analysis and quantitation of AKT, p-AKT, BAX, BCL-2 protein. Statistical analysis was performed using ANOVA followed by Dunnett’s multiple comparison test. * p < 0.05 and ** p < 0.01 indicate comparisons vs. “CTRL” group.

Article Snippet: The Annexin V-FITC/PI Detection Kit (Solarbio, China) was used to measure cell apoptosis.

Techniques: Staining, Immunofluorescence, Fluorescence, Membrane, Flow Cytometry, Microscopy, Western Blot, Quantitation Assay, Comparison

Journal: Chemical Engineering Journal

Article Title: Injectable Tumor Microenvironment-Modulated Hydrogels with Enhanced Chemosensitivity and Osteogenesis for Tumor-Associated Bone Defects Closed-Loop Management

doi: 10.1016/j.cej.2022.138086

Figure Lengend Snippet: Scheme 1. Schematics of the synthesized injectable and TME-modulated MTX-ss-MBGN GO hydrogels activated a cascade of orchestrating residual tumor apoptosis, restoring chemotherapy sensitivity and promoting bone regeneration for postoperative tumor-associated bone defects closed-loop management.

Article Snippet: To further detect cellular apoptosis treated with different particles, we used an Annexin V/PI apoptosis kit (Beyotime, Shanghai, China).

Techniques: Synthesized

Journal: Chemical Engineering Journal

Article Title: Injectable Tumor Microenvironment-Modulated Hydrogels with Enhanced Chemosensitivity and Osteogenesis for Tumor-Associated Bone Defects Closed-Loop Management

doi: 10.1016/j.cej.2022.138086

Figure Lengend Snippet: Fig. 3. MTX-ss-MBGN nanoparticles induced G1/S cell cycle arrest, ROS generation, and tumor apoptosis in UMR-106 osteosarcoma cells. (A) The cell cycle distribution of UMR-106 cells after different treatments for 72 h. (B) Analyzing the proportion of cell phases. (C) The mitochondrial membrane potential of UMR-106 cells treated with different nanoparticles for 24 h in JC-1 staining by CLSM observation. (D) The mitochondrial membrane potential of UMR-106 cells treated with different nanoparticles for 24 h or 48 h in JC-1 staining by flow cytometry. (E) Cell apoptosis was detected with Annexin V–FITC/PI double-staining using flow cytometry. (F) Statistical analysis of the apoptosis rate in different groups. (G) ROS detected with relative DCF fluorescence intensity as measured by flow cytometry. (H) Statistical analysis of relative DCF fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: To further detect cellular apoptosis treated with different particles, we used an Annexin V/PI apoptosis kit (Beyotime, Shanghai, China).

Techniques: Membrane, Staining, Flow Cytometry, Double Staining, Fluorescence

Journal: Chemical Engineering Journal

Article Title: Injectable Tumor Microenvironment-Modulated Hydrogels with Enhanced Chemosensitivity and Osteogenesis for Tumor-Associated Bone Defects Closed-Loop Management

doi: 10.1016/j.cej.2022.138086

Figure Lengend Snippet: Fig. 5. MTX-ss-MBGN GO hydrogels restrained osteosarcoma recurrence and prolonged survival days in mice by orchestrating tumor apoptosis and cell cycle exit. (A) Schematic illustrating MTX-ss-MBGN GO hydrogels treatment in a mouse model of orthotopic post-operative tumor-associated bone defects with residual tumor tissue. (B) In vivo bioluminescence imaging of K7M2 osteosarcoma tumors after surgical removal of tumors at day 21. Images of day 20 were taken before surgery. (C) Tumor growth in different groups. (D) Bodyweight changes in different groups. (E) Survival days of the mice under different treatments. (F) Histology evaluation of residual tumors under different treatments.

Article Snippet: To further detect cellular apoptosis treated with different particles, we used an Annexin V/PI apoptosis kit (Beyotime, Shanghai, China).

Techniques: In Vivo, Imaging

Journal: International Journal of Nanomedicine

Article Title: Glucose-responsive mesoporous silica nanoparticles to generation of hydrogen peroxide for synergistic cancer starvation and chemistry therapy

doi: 10.2147/IJN.S195900

Figure Lengend Snippet: ( A ) The morphological changes of HepG2 cells detected with dual staining of Hoechst 33,342/PI and captured by fluorescence microscope (magnification 200x). Arrows indicate (1) viable cells with normal nuclei; (2) live cells with apoptotic nuclei; (3) dead cells with normal nuclei; and (4) dead cells with apoptotic nuclei. ( B ) Apoptosis rate of HepG2 cells was determined by Annexin V-FITC/PI staining. ( C ) Apoptosis rate of HepG2 cells was analyzed by Annexin V-FITC/PI staining after treatment with MSNs-GOx/PLL/HA in in different concentrations (50, 100, 200, 400, 800 μg/mL) of glucose. ( D ) Change in mitochondrial membrane potential ΔΨm of HepG2 cells treated with various preparations for 8 hours analyzed by flow cytometry. Significant difference from control: * P <0.05, ** P <0.01, *** P <0.001. ( E ) Effects of treatment with different preparations on the cell cycle of HepG2 cells. ( F ) The percent of cell cycle distribution after treatment with various formulations for 24 hours. ( G ) Plasma concentration-time curves of taxol and PTX-loaded nanoparticles in rats after intravenous administration of 2.5 mg/kg PTX (mean ±SD, N=6). Abbreviations: PI, propidium iodide; FITC, fluorescein isothiocyanate; MSNs, mesoporous silica nanoparticles; GOx, glucose oxidase; PLL, poly (L-lysine); HA, hyaluronic acid.

Article Snippet: The apoptosis-inducing capability of nanoparticles was further evaluated by Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime).

Techniques: Staining, Fluorescence, Microscopy, Membrane, Flow Cytometry, Control, Clinical Proteomics, Concentration Assay