Journal: Cancers

Article Title: Curcumin-Dichloroacetate Hybrid Molecule as an Antitumor Oral Drug against Multidrug-Resistant Advanced Bladder Cancers

doi: 10.3390/cancers16173108

Figure Lengend Snippet: List of fine chemicals and assay kits used in this study.

Article Snippet: JC-1 dye , G-Biosciences (St. Louis, MO, USA) #786-1322.

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Isosteviol Protects Free Fatty Acid- and High Fat Diet-Induced Hepatic Injury via Modulating PKC-β/p66Shc/ROS and Endoplasmic Reticulum Stress Pathways

doi: 10.1089/ars.2018.7521

Figure Lengend Snippet: Effect of ISV on PA-induced mitochondrial dysfunction and oxidative stress. Rat primary hepatocytes were treated with PA (1 mM) and different concentrations of ISV (0, 1, 3, and 10 μM) for 24 h. (A) The MTP was measured by JC-1 staining followed by flow cytometry. Representative flow cytometry diagrams of three independent experiments are shown. (B) The mitochondrial mass was measured by using MitoTracker Green FM. The relative amount of each treatment group to control group was calculated. (C) Representative immunoblot images of cytosolic cytochrome c, β-actin, and Cox4i1 are shown. (D) ROS levels were measured by flow cytometry to detect intracellular fluorescence intensity of DCF. (E) Relative amounts of 4-HNE, SOD, and MDA were measured as described in the Materials and Methods section. (F) Representative immunoblot images of p66Shc in cytosol and mitochondria. Cox4i1 and Actin were used as loading control of mitochondrial and cytosolic protein, respectively. JC-1 aggregates and high DCF intensity cells ratios were calculated, respectively, based on the mean ± SEM of three independent experiments. Relative protein levels of Cytochrome c and p66Shc were calculated based on the mean ± SD of three independent experiments. Statistical significance compared with control or PA group, *p < 0.05, **p < 0.01, ***p < 0.001, and NS, no significance. Cox4i1, cytochrome c oxidase subunit 4i1; DCF, 2′,7′-dichlorofluorescein; JC-1, 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide; MTP, mitochondrial transmembrane potential; p66Shc, Src-homology-2-domain-containing transforming protein 1; ROS, reactive oxygen species; SOD, superoxide dismutase.

Article Snippet: Mitochondrial transmembrane potential Rat primary hepatocytes or HepG2 cells were treated with 1 m M PA with or without various concentrations of ISV for 24 h. The MTP was measured by using the fluorescent probe JC-1 and a BD FACSCalibur flow cytometer, as previously described ( 49 ).

Techniques: Staining, Flow Cytometry, Western Blot, Fluorescence

Journal: Antioxidants & Redox Signaling

Article Title: Isosteviol Protects Free Fatty Acid- and High Fat Diet-Induced Hepatic Injury via Modulating PKC-β/p66Shc/ROS and Endoplasmic Reticulum Stress Pathways

doi: 10.1089/ars.2018.7521

Figure Lengend Snippet: Effect of p66Shc on PA-induced hepatic injury in rat primary hepatocytes. Rat primary hepatocytes were transduced with lentiviral p66Shc shRNAs for 48 h as described in the Materials and Methods section. (A) Representative immunoblots of p66Shc and β-actin are shown. Relative protein levels of p66Shc were determined based on the mean ± SD of three independent experiments, and actin was used as an internal loading control. Statistical significance compared with control group, **p < 0.01, ***p < 0.001. (B, C) Rat primary hepatocytes were treated with 1 mM PA for 24 h after knocking down p66Shc. (B) The intracellular lipid, (C) cell apoptosis, ROS level, and MTP were measured by flow cytometry as previously described. Nile red fluorescence intensity, Annexin V positive cells ratio, high DCF intensity cells ratio, and JC-1 aggregates were calculated, respectively, based on the mean ± SEM of three independent experiments. (D, E) Rat primary hepatocytes were transduced with lentiviral p66Shc or p66Shc (S36D) phosphomimetic mutation for 24 h as described in the Materials and Methods section, and then they were treated with ISV (0, 1, 3, or 10 μM) for 24 h. (D) Intracellular ROS levels were measured, and high DCF intensity cells ratio was calculated based on the mean ± SEM of three independent experiments. (E) The relative amount of LDH released to culture medium represents the mean ± SD of three independent experiments. *p < 0.05, ***p < 0.001, and NS, no significance. C-shRNA, control shRNA. Color images are available online.

Article Snippet: Mitochondrial transmembrane potential Rat primary hepatocytes or HepG2 cells were treated with 1 m M PA with or without various concentrations of ISV for 24 h. The MTP was measured by using the fluorescent probe JC-1 and a BD FACSCalibur flow cytometer, as previously described ( 49 ).

Techniques: Transduction, Western Blot, Flow Cytometry, Fluorescence, Mutagenesis, shRNA

Journal: Antioxidants & Redox Signaling

Article Title: Isosteviol Protects Free Fatty Acid- and High Fat Diet-Induced Hepatic Injury via Modulating PKC-β/p66Shc/ROS and Endoplasmic Reticulum Stress Pathways

doi: 10.1089/ars.2018.7521

Figure Lengend Snippet: Effect of PKC-β on PA-induced hepatic injury in rat primary hepatocytes. Rat primary hepatocytes were transduced with lentiviral PKC-β shRNAs for 48 h as described in the Materials and Methods section. (A) Representative immunoblots of PKC-β and β-actin are shown. Relative protein levels of PKC-β were determined based on the mean ± SD of three independent experiments, and β-actin was used as an internal loading control. Statistical significance compared with control group, **p < 0.01, ***p < 0.001. (B–D) Rat primary hepatocytes were further treated with PA (1 mM) with or without ISV (10 μM) or RBX (20 nM) for 24 h. (B) The intracellular lipid, (C) cell apoptosis, ROS levels, and MTP were measured by flow cytometry as previously described. Nile red fluorescence intensity, Annexin V positive cells ratio, high DCF intensity cells ratio, and JC-1 aggregates were calculated, respectively, based on the mean ± SEM of three independent experiments. Statistical significance compared with control-shRNA or PA group, *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Representative immunoblot images of p66Shc in cytosol and mitochondria are shown. Cox4i1 and β-actin were used as internal loading controls for mitochondria and cytosol, respectively. Relative protein levels of p66Shc represent the mean ± SD of three independent experiments. Statistical significance compared with control-shRNA or PA group, *p < 0.05, **p < 0.01. (E) Rat primary hepatocytes were treated with 50 nM dPPA with or without PA and ISV (0, 1, 3, and 10 μM) for 24 h. Hepatocyte apoptosis was measured by flow cytometry. Annexin V positive cells ratio was calculated based on the mean ± SEM of three independent experiments. Statistical significance compared with PA group, *p < 0.05, **p < 0.01, ***p < 0.001, and NS, no significance. dPPA, 12-deoxyphorbol 13-phenylacate 20-acetate. Color images are available online.

Article Snippet: Mitochondrial transmembrane potential Rat primary hepatocytes or HepG2 cells were treated with 1 m M PA with or without various concentrations of ISV for 24 h. The MTP was measured by using the fluorescent probe JC-1 and a BD FACSCalibur flow cytometer, as previously described ( 49 ).

Techniques: Transduction, Western Blot, Flow Cytometry, Fluorescence, shRNA