Journal: Antioxidants

Article Title: Development of an AAPH-Induced Oxidative Stress Model in Bovine Mammary Epithelial Cells and Investigation of Its Molecular Mechanisms

doi: 10.3390/antiox15040460

Figure Lengend Snippet: ( A ) Representative fluorescence images of intracellular ROS levels detected using the DCFH-DA probe. Green fluorescence intensity reflects the relative level of reactive oxygen species. Scale bar = 50 μm. ( B ) Quantitative analysis of relative ROS fluorescence intensity. Data are presented as mean ± SD from three independent experiments. NC: negative control (untreated cells); PC: positive control (cells treated with the ROS-positive control reagent provided in the kit); AAPH: cells treated with 1000 μmol/L AAPH for 6 h; NAC: cells pretreated with N-acetylcysteine prior to AAPH treatment. ( C ) Quantitative analysis of the relative ratio of red (J-aggregates) to green (JC-1 monomer) fluorescence intensity measured by the JC-1 assay. A decrease in this ratio indicates loss of mitochondrial membrane potential. (ns: p > 0.05; ** p < 0.01; and *** p < 0.001). ( D ) Representative fluorescence images of JC-1 staining. Red fluorescence indicates JC-1 aggregates in mitochondria with high membrane potential (healthy mitochondria), while green fluorescence indicates JC-1 monomers in mitochondria with low membrane potential (mitochondrial injury). The merged images show colocalization. Scale bar = 50 μm.

Article Snippet: AAPH, N-Acetylcysteine (NAC), the Cell Counting Kit-8, ROS Assay Kit, and JC-1 Mitochondrial Membrane Potential Assay Kit were purchased from MedChemExpress (MCE) (Monmouth Junction, NJ, USA).

Techniques: Fluorescence, Negative Control, Positive Control, Membrane, Staining

Journal: Bioactive Materials

Article Title: Activatable companion theranostics for dual-modality imaging-escorted pyroptosis-propelled synergistic cancer therapy

doi: 10.1016/j.bioactmat.2025.08.036

Figure Lengend Snippet: In vitro mechanism of synergistic therapy mediated by LET-Cl@GOx. Changes of a extracellular acidification rate (ECAR) and b relevant glycolysis ability after treatments. Variations of c mitochondrial oxygen consumption rate (OCR) and d their associated quantifications. e Measurements of ATP production rate with various treatments. f Brightfield images of A549 cells following different treatments. Scale bar: 50 μm g FL images of cells stained with Annexin V-FITC/PI after various treatments. Scale bar: 50 μm h Immunofluorescence staining images of caspase-3 activation. Scale bar: 50 μm. i Western blotting analysis of indicated protein expression following treatments. j Measurements of LDH release after indicated treatments. k The process of pyroptosis through the Caspase-3/GSDME pathway induced by LET-Cl@GOx (created with BioRender.com ). The data are presented as the means ± SDs, n = 3. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. The symbols “+” indicate laser irradiation (808 nm, 0.6 W cm −2 ).

Article Snippet: Afterwards, the cells were stained Mitochondrial Membrane Potential Assay Kit (Beyotime, China).

Techniques: In Vitro, Staining, Immunofluorescence, Activation Assay, Western Blot, Expressing, Irradiation

Journal: The Chinese journal of physiology

Article Title: A basal level of γ-linolenic acid depletes Ca 2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells.

doi: 10.4103/cjp.cjp_30_21

Figure Lengend Snippet: Figure 3: Effect of gamma‑linolenic acid on mitochondrial membrane potential. BT‑474 cells were treated with 30 µM gamma‑linolenic acid for different time periods and mitochondrial membrane potential was quantified by JC‐1 fluorescence assay. Red emission/green emission ratio lower than control values indicates depolarization. Results are mean ± standard error of mean from three independent experiments. *Significantly (P < 0.05) different from control.

Article Snippet: Measurement of mitochondrial membrane potential Mitochondrial membrane potential was measured using a Mitochondrial Membrane Potential Assay Kit (#12664; Cell Signaling, Danvers, MA, USA).

Techniques: Membrane, Fluorescence, Control

Journal: The Chinese journal of physiology

Article Title: A basal level of γ-linolenic acid depletes Ca 2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells.

doi: 10.4103/cjp.cjp_30_21

Figure Lengend Snippet: Figure 4: Effect of gamma‑linolenic acid on mitochondrial Ca2+. BT‑474 cells were treated with 30 µM gamma‑linolenic acid for different time periods and mitochondrial Ca2+ level was quantified by flow cytometry using Rhod 2 as fluorescence probe. Results are mean ± standard error of mean from three independent experiments. *Significantly (P < 0.05) different from control.

Article Snippet: Measurement of mitochondrial membrane potential Mitochondrial membrane potential was measured using a Mitochondrial Membrane Potential Assay Kit (#12664; Cell Signaling, Danvers, MA, USA).

Techniques: Flow Cytometry, Fluorescence, Control

Journal: Scientific Reports

Article Title: Metabolic injury-induced NLRP3 inflammasome activation dampens phospholipid degradation

doi: 10.1038/s41598-017-01994-9

Figure Lengend Snippet: Mitochondrial damage and loss of tubular functional properties. ( A ) Mitochondria membrane permeabilization (MMP) assessed with MMP MITO-ID® Membrane Potential Detection cationic dye that fluoresces either green (as monomer in the cytosol) or orange (as aggregate in the mitochondria) depending upon membrane potential status. MMP indicated by increased ratio of %FITC + PE −/%FITC + PE + HK2 cells, quadrants Q3/Q2 of the scatterplot (FC). Data normalized to Ctr ( = 1). ( B ) Oxidative stress detected by Green fluorescent ROS Detection Reagent (FC). Data normalized by subtracting Ctr MFIs. Histogram plotting the FITC picks of HK2 cells exposed to LPDS (green), LDL (blue) and oxLDL (red). ( C ) Transmission electron microscope images of kidney sections derived from mice fed a Western-diet. Arrows indicate damaged (D) and normal (N) mitochondria. Scale bars, 2 and 1 µm. ( D ) Uptake of green fluorescent deoxyglucose analog (2-NBDG) by MDCK cells (FC). Control MFIs subtracted from n/oxLDL MFIs, negative values indicative of a reduction in 2-NBDG uptake. ( E ) Luminescent ATP Detection in MDCK and ( F ) HK2 cells. Data shown as ratio luminescence unit (LU)/µg protein of cell lysates divided by the Ctr values (Ctr = 1). ( G , H ) Westernblot for SGLT2 using protein lysates of HK2 cells after ( G ) 5 or ( H ) 3 days treatment. β-actin used as loading control. SGLT2 expression normalized to Ctr ( = 1). ( A , B , H ) Assays at day 3, ( D – G ) day 5. In dotplot graphs, each dot represents the average of one independent experiment; mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For detection of alterations in lysosomal and mitochondrial organelles and tubular absorption, the following reagents were used: HCS LipidTOX™ Red phospholipidosis detection reagent (1:2000, Thermo Fisher Scientific), 50 nM LysoTracker Red DND-99 (Thermo Fisher Scientific), 1 µM pH-sensitive LysoSensor Green DND-189 (Thermo Fisher Scientific), 50 µg/ml FITC-Dextran 10/40 KDa (Sigma-Aldrich), 1 µM Fluo-4-AM (Invitrogen), MITO-ID® Membrane Potential Detection Kit (Enzo Life Sciences) and Oxidative Stress Detection Reagent (Enzo Life Sciences), 5 mM 2-NBDG (Cayman Chemical), 0,1 µg/ml Nile Red (Sigma Aldrich).

Techniques: Functional Assay, Transmission Assay, Microscopy, Derivative Assay, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Metabolic injury-induced NLRP3 inflammasome activation dampens phospholipid degradation

doi: 10.1038/s41598-017-01994-9

Figure Lengend Snippet: Negative regulation of SIRT1/LKB1/AMPK pathway by NLRP3/ASC/CASP1 immunocomplex. ( A ) Westernblots showing the expression of SIRT1, phosphorylated and total AMPK and LKB1 in HK2 cell lysates. Control value equal to 1 after normalization; β-actin used as loading control. ( B ) Use of SIRT1 activator (SRT1720) and AMPK activators (AICAR and resveratrol) to reduce phospolipidosis in respect to untreated cells after LDL loading (FC). Data shown as differences in MFI values. ( C ) Effects of SIRT1 knockdown (shRNA), overexpression (expression plasmid) of SIRT1 and LKB1 on the rate of endolysosomal phospholipid content in HK2 TEC in respect to their respective controls (non-targeting shRNA, empty vector). Data shown as differences in MFI values (FC). ( D ) Westernblot showing the activation rate of AMPK and LKB1 in LDL-loaded HK2 cells stably expressing shRNA targeting SIRT1/NLRP3 or non-targeting shRNA (shNT); β-actin used as loading control. Controls (shNT = 1) used for normalization. ( E ) Phospholipid storage in HK2 TEC expressing shRNA for NLRP3 / SIRT1 gene silencing (FC). MFI values of non-targeting shRNA expressing cells subtracted from all MFIs. ( F ) Oxidative stress detected by Green fluorescent ROS Detection Reagent (FC). Differences in MFI values of HK2 cells expressing shRNA targeting NLRP3 as compared to cells expressing shNT. ( F ) Mitochondria damage assessed with the MMP MITO-ID® assay; variations of MMP indicated by increased ratio of %FITC + PE-/%FITC + PE + HK2 cells (FC). Data normalized to values of cells expressing shNT ( = 1). ( B , C , E , F , G ) Dots representing averages of independent experiments. Mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For detection of alterations in lysosomal and mitochondrial organelles and tubular absorption, the following reagents were used: HCS LipidTOX™ Red phospholipidosis detection reagent (1:2000, Thermo Fisher Scientific), 50 nM LysoTracker Red DND-99 (Thermo Fisher Scientific), 1 µM pH-sensitive LysoSensor Green DND-189 (Thermo Fisher Scientific), 50 µg/ml FITC-Dextran 10/40 KDa (Sigma-Aldrich), 1 µM Fluo-4-AM (Invitrogen), MITO-ID® Membrane Potential Detection Kit (Enzo Life Sciences) and Oxidative Stress Detection Reagent (Enzo Life Sciences), 5 mM 2-NBDG (Cayman Chemical), 0,1 µg/ml Nile Red (Sigma Aldrich).

Techniques: Expressing, shRNA, Over Expression, Plasmid Preparation, Activation Assay, Stable Transfection