Journal: Cells

Article Title: Oxidative Stress-Induced Unscheduled CDK1–Cyclin B1 Activity Impairs ER–Mitochondria-Mediated Bioenergetic Metabolism

doi: 10.3390/cells10061280

Figure Lengend Snippet: Induction of the formation of phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)–cyclin B1 complexes by apigenin. Nasopharyngeal carcinoma (NPC) cells were treated with vehicle (−) or apigenin (35 μM) for 36 h. ( A ) The levels of the indicated proteins in the total cell (T) lysates were determined using Western blot analysis with specific antibodies. β-Actin was used as an internal control for sample loading. ( B ) The antibody used for coimmunoprecipitation is indicated at the top. The proteins from the immunoprecipitated complexes were detected using Western blotting with specific antibodies. Normal IgG was used as a control for antibody specificity.

Article Snippet: ASM shRNA phasmid, CDK1 siRNA, cyclin B1 siRNA, control siRNA, and western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Control, Immunoprecipitation

Journal: Cells

Article Title: Oxidative Stress-Induced Unscheduled CDK1–Cyclin B1 Activity Impairs ER–Mitochondria-Mediated Bioenergetic Metabolism

doi: 10.3390/cells10061280

Figure Lengend Snippet: Apigenin-induced phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)–cyclin B1 activity involved in dysregulation of the endoplasmic reticulum (ER)/mitochondria bioenergetic metabolism. ( A – E ) At 12 h after transfection with control small interfering RNA (siRNA), cyclin B1 siRNA, or CDK1 siRNA, NPC cells were treated with vehicle (-), apigenin (35 μM), Ro-3306 (3 μM), or apigenin (35 μM), and Ro-3306 (3 μM) for 36 h. The levels of the indicated proteins in the whole cell lysates of transfected cells were determined by Western blot analysis with specific antibodies. The levels of cytosolic calcium (Ca ++ ) and reactive oxygen species (ROS) were determined by measuring the increased fluorescence of Indo-1 and 2,7-dichlorodihydrofluorescein by flow cytometry. The values of glucose and adenosine triphosphate (ATP) were analyzed using glucose assay and ATP-based CellTiter-Glo Luminescent Cell Viability kits, respectively. The values are presented as the means ± standard error of three independent experiments. * p < 0.05: significantly different from vehicle- or apigenin-treated cells.

Article Snippet: ASM shRNA phasmid, CDK1 siRNA, cyclin B1 siRNA, control siRNA, and western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Transfection, Control, Small Interfering RNA, Western Blot, Fluorescence, Flow Cytometry, Glucose Assay

Journal: Cells

Article Title: Oxidative Stress-Induced Unscheduled CDK1–Cyclin B1 Activity Impairs ER–Mitochondria-Mediated Bioenergetic Metabolism

doi: 10.3390/cells10061280

Figure Lengend Snippet: Apigenin-induced oxidative stress-dependent lipid raft-associated acid sphingomyelinase (ASM) activity causes dysregulation of the endoplasmic reticulum (ER)/mitochondria bioenergetic and lipid metabolism. ( A , B , E – M ) At 12 h after transfection with an empty vector, GFP shRNA or ASM shRNA cells were treated with either vehicle (−), apigenin (35 μM), 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) (15 nmole), C2-ceramide (6 μM), imipramine (5 μM), apigenin (35 μM) plus imipramine (5 μM), N-acetyl-L-cysteine (NAC) (100 μM), or apigenin (35 μM) and NAC (100 μM) for 36 h. The levels of the indicated proteins in the cell lysates were determined by Western blot analysis with specific antibodies. β-Actin was used as an internal control for sample loading. Detergent-resistant membranes (DRM) fractions were prepared by flotation on a sucrose density gradient. The antibodies used for co-immunoprecipitation are indicated at the top of the figure. The proteins in the immunoprecipitated complexes from the DRM were analyzed by Western blot using specific antibodies. ASM activities were determined by Acidic Sphingomyelinase Assay Kit. The lipids were extracted from the DRM fractions, and cholesterol, sphingomyelin, and ceramide were quantitated by Amplex Red Cholesterol Assay Kit and thin-layer chromatography, respectively. Ceramide concentrations were normalized to phospholipid phosphate. The generation of cytosolic level of calcium (Ca ++ ) and reactive oxygen species (ROS) were monitored by measuring the increased fluorescence of Indo-1 and 2,7-dichlorodihydrofluorescein by flow cytometry. The values of glucose and adenosine triphosphate (ATP) were analyzed using glucose assay and ATP-based CellTiter-Glo Luminescent Cell Viability kits, respectively. Cell viability was determined by the flow cytometric analysis of PI uptake. The values are presented as the means ± standard error of three independent experiments. * p < 0.05: significantly different from vehicle- or apigenin-treated cells. ( C ) Cells were treated with vehicle (−), apigenin (35 μM), or apigenin (35 μM) and NAC (100 μM) for 36 h. DRM and detergent-soluble (DS) fractions were prepared by flotation on a sucrose density gradient. The levels of the indicated proteins in the lysates of vehicle- or apigenin-treated DRM and DS fractions were determined by Western blot analysis using specific antibodies. ( D ) Cells were treated with vehicle (−), apigenin (35 μM), or apigenin (35 μM) and NAC (100 μM). Cells were harvested 36 h after treatment with vehicle or apigenin, and cell pellets were resuspended in hypotonic buffer. Crude homogenates were incubated with 5 mM bismaleimidohexane (BMH) in PBS for 30 min at room temperature and then subjected to subcellular fractionation to obtain the mitochondrial (Mt) and ER/microsomal (Ms) fractions. In total, 20 μg of total protein from the recovered fractions was analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and probed with specific antibodies, as indicated. Cox-2, calnexin, and α-tubulin were used as internal controls for the mitochondria, ER, and cytosol, respectively.

Article Snippet: ASM shRNA phasmid, CDK1 siRNA, cyclin B1 siRNA, control siRNA, and western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Transfection, Plasmid Preparation, shRNA, Western Blot, Control, Immunoprecipitation, Amplex Red Cholesterol Assay, Thin Layer Chromatography, Fluorescence, Flow Cytometry, Glucose Assay, Incubation, Fractionation, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: Cells

Article Title: Oxidative Stress-Induced Unscheduled CDK1–Cyclin B1 Activity Impairs ER–Mitochondria-Mediated Bioenergetic Metabolism

doi: 10.3390/cells10061280

Figure Lengend Snippet: A molecular model for the attenuation of endoplasmic reticulum (ER)−mitochondria-mediated bioenergetic and lipid metabolic homeostasis by apigenin in nasopharyngeal carcinoma (NPC) cells. ( A ) The formation of clustered phosphatidylinositol 3-kinase (PI3K)−protein kinase B (Akt)−GTP-ras-related C3 botulinum toxin substrate 1 (Rac1) signaling platform in the lipid raft membranes constitutes a central element in the control of p21-mediated cyclin dependent kinase 1 (CDK1)−cyclin B1 inactivation to contribute initiation of B-cell lymphoma-extra large (BCL-x L )/ B cell lymphoma 2 (BCL-2)-inhibited Bcl-2-associated x protein (BAX)/Bcl-2 antagonist killer 1 (BAK) pro-apoptotic and metabolic activities, thereby modulates the ER−mitochondria-regulated bioenergetic, redox, and calcium (Ca ++ ) homeostasis. ( B ) Under the condition of cellular apigenin uptake, apigenin induces the lipid raft membrane translocalization of acid sphingomyelinase (ASM) from lysosome to promote the generation of ceramide-enriched lipid raft membranes by triggering oxidative stress-induced ER Ca ++ release, thus inducting the formation of p110α-free homodimerized p85α–unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 (PTEN) tetrameric complexes and thereby disturbing the interaction between p85α and p110α in the lipid raft membranes. The resultant lipid raft membrane-associated p85α–PTEN complexes can negatively regulate Akt activity and attenuate Rac1 activation by dephosphorylating the 3-position of phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) to phosphatidylinositol-4,5-bisphosphate (PIP 2 ). Akt inactivation was accompanied by induction of caspase-3-mediated p21 cleavage resulting the mitochondrial localization of p-CDK1 (Thr 161)–cyclin B1 complexes, which causes the phosphorylation of ER–mitochondria-associated BCL-x L (Ser 62)/BCL-2 (Thr 69/Ser 87) and then results in the dimerization/oligomerization of BAX/BAK, thereby attenuating the regulatory effects of BCL-x L /BCL-2 on the control of ER−mitochondria-regulated bioenergetic, redox, and Ca ++ homeostasis as well as resulting in apoptotic cell death of NPC cells.

Article Snippet: ASM shRNA phasmid, CDK1 siRNA, cyclin B1 siRNA, control siRNA, and western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Membrane, Activity Assay, Activation Assay, Phospho-proteomics