Journal: Journal of Cellular and Molecular Medicine

Article Title: Synergistic cytotoxicity of perifosine and ABT ‐737 to colon cancer cells

doi: 10.1111/jcmm.17636

Figure Lengend Snippet: The effect of tumour environment on the cytotoxicity of perifosine and ABT‐737 to HT‐29 cells in monolayers. (A–C,E,F) Cells were pretreated in different tumour environments for 72 h and then treated with perifosine or ABT‐737 for 48 h. (A) Cytotoxicity was assessed by MTT. Results are presented as mean ± SD. (B) The isobolograms of perifosine and ABT‐737 in constant and non‐constant drug ratios in all environments tested. The diagonal line indicates additivity. Data points below the additivity line indicate synergy, data points above indicate antagonism. (C) Fa–CI plot of perifosine and ABT‐737 in constant drug ratio (1:1) in all tested tumour microenvironments. CI was plotted on the y‐axis as a function of efficacy (Fa) on the x‐axis. Drug dose combinations are shown in the table. (D) Cells were pretreated in different tumour environments for 72 h, then treated with perifosine/ABT‐737 for 24 h, and cytotoxicity was determined by ATP assay. Results are presented as mean of ATP level per well ± SD. (E) Mitochondrial membrane potential was measured as JC‐1 probe fluorescence using flow cytometry. (F) ROS level was assessed using DCFH‐DA staining and flow cytometry. Data are presented as mean ± SD. All the experiments were repeated at least three times. Significant differences between treatments were determined by t ‐test; * p < 0.05, ** p < 0.01, *** p < 0.001 for controls and induced cells (A,E,D) or for the similarly treated samples in NaL NORMOXIA and LA NORMOXIA (F) and # p < 0.05, ## p < 0.01, ### p < 0.001 for single drug and combination‐induced cells, unless otherwise indicated.

Article Snippet: Cells in 2D were adapted to culture media with different pH and oxygen values for 72 h and then treated with 5 μM perifosine, 5 μM ABT‐737 and their combination for 24 h. For spheroid induction, chemical doses were raised to a concentration of 20 μM and the exposure time was increased to 48 h. Quantification of cellular ATP was performed using the ATP assay kit (Cayman Chemicals, Ann Arbor, Michigan, USA) as previously described.,

Techniques: ATP Assay, Membrane, Fluorescence, Flow Cytometry, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: A piperazidine derivative of 23-hydroxy betulinic acid induces a mitochondria-derived ROS burst to trigger apoptotic cell death in hepatocellular carcinoma cells

doi: 10.1186/s13046-016-0457-1

Figure Lengend Snippet: ρ0 HepG2 cells were less sensitive to B5G9 treatment. a Mitochondrial complex activities of wt HepG2 or ρ0 HepG2 were detected by related assay kits, *** P ≤ 0.001. b ATP production of ρ0 HepG2 cells was significantly decreased in the present of 2-DG. After being treated with 2-DG (2 mM) for 2 h, ATP level of wt HepG2 or ρ0 HepG2 was measured by a CellTiter-Glo Luminescent Assay, *** P ≤ 0.001. c ρ0 HepG2 had a lower ATP synthetase activity than wt HepG2. The ATP synthetase activity of wt HepG2 treated in the present or absent of oligomycin (6 μM) and ρ0 HepG2 was measured by a cayman ATP synthetase activity kit, ** P ≤ 0.01. d ρ0 HepG2 cells had no significant change in mitoSOX red fluorescence after B5G9 treatment, original magnifications : 630 ×; scale bar: 10 μm. e ρ0 HepG2 cells were less sensitive to B5G9 treatment. Being treated with various concentrations of B5G9 for 12 h, cell viability was measured by MTT assay. * P ≤ 0.01, *** P ≤ 0.001

Article Snippet: The ATP synthetase activity of wt HepG2 treated in the present or absent of oligomycin (6 μM) and ρ0 HepG2 was measured by a cayman ATP synthetase activity kit, ** P ≤ 0.01. d ρ0 HepG2 cells had no significant change in mitoSOX red fluorescence after B5G9 treatment, original magnifications : 630 ×; scale bar: 10 μm. e ρ0 HepG2 cells were less sensitive to B5G9 treatment.

Techniques: Luminescence Assay, Activity Assay, Fluorescence, MTT Assay

Journal: JCI Insight

Article Title: Mitochondrial Ca 2+ -coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome

doi: 10.1172/jci.insight.176212

Figure Lengend Snippet: ( A ) Structural representation of the Kir6.2/SUR1 K ATP channel (PDB ID: 5WUA) with homologous positions of Kir6.1 [V65M] and SUR2 [A476V] mutations in mice indicated. ( B ) Schematic diagram illustrating K ATP channel activation by the synthetic opener pinacidil (Pin) and inhibition by the sulfonylurea compound glibenclamide (Glib). ( C ) Representative whole-cell voltage-clamp recordings from acutely isolated mesenteric vascular SMCs from WT and Kir6.1 wt/VM mice. ( D ) Summary data of whole-cell current densities from voltage-clamp recordings of WT and Kir6.1 wt/VM SMCs showing significant increases in basal and pinacidil-activated K ATP conductance, which are resistant to glibenclamide in Kir6.1 wt/VM Data are presented as means ± SEM ( n = 5 cells from 3 animals per group; * P < 0.05, 2-way ANOVA with Tukey’s post hoc test). ( E ) Representative whole-cell voltage-clamp recordings of freshly isolated mesenteric vascular endothelial cells from WT and Kir6.1 wt/VM mice. ( F ) Summary data of whole-cell current densities from voltage-clamp recordings of vascular endothelial cells from WT and Kir6.1 wt/VM with indicated treatments. Data are presented as means ± SEM ( n = 4–5 cells from 3 animals per group; * P < 0.05, 2-way ANOVA with Tukey’s post hoc test). ( G ) Representative whole-cell current-clamp recordings from freshly isolated mesenteric vascular endothelial cells isolated from WT and Kir6.1 wt/VM mice. Recordings were recorded initially under basal conditions and then following treatment with pinacidil and glibenclamide. ( H ) Summary of current-clamp recordings. Data are presented as means ± SEM ( n = 5–7 cells from 3 animals per group; * P < 0.05, 2-way ANOVA with Tukey’s post hoc test).

Article Snippet: In some experiments, arteries were treated with the K ATP channel activator pinacidil (10 μM; Cayman Chemical) and/or the K ATP channel blocker glibenclamide (10 μM; Tocris Bioscience).

Techniques: Activation Assay, Inhibition, Isolation

Journal: JCI Insight

Article Title: Mitochondrial Ca 2+ -coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome

doi: 10.1172/jci.insight.176212

Figure Lengend Snippet: ( A ) Representative grayscale and pseudocolored images of mesenteric arteries from Cdh5 -GCaMP8 mice mounted en face. Recordings were made initially under baseline conditions for 50 seconds and then in the presence of CCh (10 μM) for 150 seconds, followed by treatment with pinacidil (Pin; 10 μM) for 150 seconds and subsequently with glibenclamide (Glib; 10 μM) in the presence of CCh and pinacidil for 150 seconds. Colored boxes show regions of interest (ROIs) where Ca 2+ events occurred. The colors indicate the different ROIs that are represented in the time series traces. Scale bar: 10 μm. ( B ) Representative ΔF/F 0 versus time plots of Ca 2+ events from multiple Ca 2+ -event sites. ( C ) Summary data showing the effects of pinacidil and glibenclamide on the amplitude (ΔF/F 0 ), frequency (Hz), and number of active sites. Data are presented as means ± SEM ( n = 6 arteries from 3 animals; * P < 0.05, 1-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: In some experiments, arteries were treated with the K ATP channel activator pinacidil (10 μM; Cayman Chemical) and/or the K ATP channel blocker glibenclamide (10 μM; Tocris Bioscience).

Techniques:

Journal: JCI Insight

Article Title: Mitochondrial Ca 2+ -coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome

doi: 10.1172/jci.insight.176212

Figure Lengend Snippet: ( A ) Representative grayscale and pseudocolored images of mesenteric arteries from Cdh 5-GCaMP8 and Cdh 5-GCaMP8 x Kir6.1 wt/VM mice mounted en face. Recordings were made initially under baseline conditions for 50 seconds and then in the presence of CCh (10 μM) for 150 seconds, followed by treatment with pinacidil (Pin; 10 μM) for 150 seconds and subsequently with glibenclamide (Glib; 10 μM) in the presence of CCh and pinacidil for 150 seconds. Colored boxes show ROIs with active Ca 2+ signals. The colors indicate the different ROIs that are represented in the time series traces. Scale bar: 10 μm. ( B ) Representative ΔF/F 0 versus time plots of Ca 2+ events from multiple ROIs. ( C–E ) Summary data showing the effects of pinacidil and glibenclamide on the amplitude (ΔF/F 0 ), frequency (Hz), and number of active sites. Data are presented as means ± SEM ( n = 6 arteries from 3 animals per group; * P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: In some experiments, arteries were treated with the K ATP channel activator pinacidil (10 μM; Cayman Chemical) and/or the K ATP channel blocker glibenclamide (10 μM; Tocris Bioscience).

Techniques:

Journal: JCI Insight

Article Title: Mitochondrial Ca 2+ -coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome

doi: 10.1172/jci.insight.176212

Figure Lengend Snippet: ( A ) Representative images of mesenteric arteries from WT and Kir6.1 wt/VM mice mounted en face and loaded with the mitochondrial-specific Ca 2+ indicator X-Rhod-1, AM. Changes in X-Rhod-1 fluorescence were detected following treatment with CCh (10 μM), pinacidil (Pin; 10 μM), and glibenclamide (Glib; 10 μM). Boxes show regions of interest (ROIs). Scale bar: 10 μm. ( B ) Representative ΔF/F 0 versus time plots of X-Rhod-1 fluorescence for each condition. Arrows indicate application of the agonist. ( C ) Summary data showing changes in X-Rhod-1 fluorescence intensity. Data are presented as means ± SEM ( n = 9 arteries from 3 animals per group; * P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test). ( D ) Representative images of mesenteric arteries from WT and Kir6.1 wt/VM mice mounted en face and loaded with the superoxidesensitive fluorescent dye CellRox. Changes in CellRox fluorescence were imaged following treatment with CCh, (10 μM), Pin (10 μM), PEG-SOD (100 U/mL), the mitochondrial ROS scavenger mitoTEMPO (5 μM), or a combination of SOD (500 U/mL) and catalase (500 U/mL). Boxes show regions of interest (ROIs). Scale bar: 10 μm. ( E ) Representative ΔF/F 0 versus time plots of the change in fluorescence intensity under each condition. Arrows indicate application of the agonist. ( F ) Summary data showing changes in CellRox fluorescence intensity. Data are presented as means ± SEM ( n = 9 arteries from 3 animals per group; * P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: In some experiments, arteries were treated with the K ATP channel activator pinacidil (10 μM; Cayman Chemical) and/or the K ATP channel blocker glibenclamide (10 μM; Tocris Bioscience).

Techniques: Fluorescence

Journal: JCI Insight

Article Title: Mitochondrial Ca 2+ -coupled generation of reactive oxygen species, peroxynitrite formation, and endothelial dysfunction in Cantú syndrome

doi: 10.1172/jci.insight.176212

Figure Lengend Snippet: ( A ) Representative recordings and ( B ) summary data showing CCh-evoked, endothelium-dependent dilation of mesenteric arteries from Kir6.1 wt/VM mice before and after treatment with the membrane-permeable intracellular ROS scavenger PEG-SOD (100 U/mL). Data are presented as means ± SEM ( n = 5 vessels from 4 animals per group; * P < 0.05, 2-way ANOVA with Šídák’s multiple comparisons test). ( C ) Representative recordings and ( D ) summary data showing CCh-evoked, endothelium-dependent dilation of mesenteric arteries from Kir6.1 wt/VM mice before and after treatment with the mitochondria-targeted superoxide mimetic mitoTEMPO (5 μM). Data are presented as means ± SEM ( n = 6 vessels from 4 animals per group; * P < 0.05, 2-way ANOVA with Šídák’s multiple comparisons test). ( E ) Schematic depicting the proposed model for endothelial dysfunction associated with Cantú syndrome. Endothelial cell K ATP GOF hyperpolarizes the plasma membrane, increasing the electrochemical driving force for Ca 2+ influx. Elevated cytosolic Ca 2+ levels are partially buffered by the mitochondria, leading to increased ROS generation. Elevated mitochondrial ROS levels reduce ·NO bioavailability and generate ONOO – in situ, impairing endothelial-dependent vasodilation. Secondarily, disruption of mitochondrial and cell homeostasis promotes cytosolic ONOO- formation and further affects vascular function and integrity ( , , ).

Article Snippet: In some experiments, arteries were treated with the K ATP channel activator pinacidil (10 μM; Cayman Chemical) and/or the K ATP channel blocker glibenclamide (10 μM; Tocris Bioscience).

Techniques: Membrane, Clinical Proteomics, In Situ, Disruption