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

Journal: PLoS ONE

Article Title: Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor

doi: 10.1371/journal.pone.0036418

Figure Lengend Snippet: Cells were treated with berberine at indicated concentrations for 18 h (A and B), as described in . Intracellular ROS levels were detected using DHE probe and the amount of fluorescence was measured using flow cytometry. The fold change of the amount of fluorescence was calculated by comparing that in the treated groups to the control group (A). Cells were treated with the JC-1 probe and then analyzed by flow cytometry to measures the fraction of cells with polarized or depolarized mitochondrial membrane (B). Cells were treated with berberine at 50 µM for 18 h in the presence or absence of 5 µM of Tiron, a ROS scavenger. Cell number change (C) and LDH release (D) were detected as described in . * p <0.01 compared to the control group, # p <0.01 compared to the berberine treated group.

Article Snippet: Δ ψ was measured using the Flow Cytometry Mitochondrial Membrane Potential Detection kit (BD™ Mitoscreen) according to the manufacturer’s instructions.

Techniques: Fluorescence, Flow Cytometry