Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Appendix 3.1.1: Constitution ‘antioxidant cocktail’The antioxidant cocktail that will be used will consist of the following antioxidants, in the relativeconcentrations indicated (Table B.2). These concentrations are based on concentrations usually effectivein lowering ROS. Equal volumes of the various antioxidants in the concentrations indicated will form thecocktail, so that it will contain all antioxidants in the relative concentrations indicated. To determine therequired dose for lowering ROS levels, the cocktail will be further diluted in PBS to various extents (1x to10.000x).Antioxidant Effect Concentration Relative concentrationTempol SOD mimetic 2,5 mM 200Diethyldithiocarbamic SOD mimetic 50 µM 4acid (DDC)N-acetyl-L-cysteine Increases glutathione 5 mM 400(NAC)poolsEUK-8 (salenmanganeseSOD/catalase mimetic 12,5 µM 1complex)Table B.2 Overview Antioxidant cocktailMaterials:4. 4-hydroxy-TEMPO (tempol) (Sigma)5. Diethyldithiocarbamic acid, lead salt, moist (DDC) (Sigma)6. N-acetyl-L-cysteine (NAC) (Sigma)7. EUK-8 (Eukarion, Inc.)Appendix 3.1.2: Increasing ROS levelsH 2 O 2 in PBS will be added to the medium in concentrations varying from 10 µM to 20 mM.Antimycin A, ATP and Diazoxide will be added to the medium in the concentrations indicated in the text.Materials:6. H 2 O 2 in PBS (Sigma)7. Antimycin A (Sigma)8. Adenosine 5’-triphosphate, Disodium salt (Sigma)9. Diazoxide (Sigma)Appendix 3.1.3: Northern blotMaterials:• PrimerBank database ()• Massachusetts General Hospital DNA Core Facility• RNEasy kit (QIAGEN)• NorthernMax (AM1940) (Ambion)Appendix 3.1.4: Real time RT-PCRThe following kits will be used for RNA isolation and two-step real time RT-PCR. SYBR Green is a dyethat selectively binds to double-stranded DNA.• For RNA purification: RNEasy kit (QIAGEN)• For reverse transcription: QuantiTect Reverse Transcriptase Kit (QIAGEN)• For PCR of the target transcript: QuantiTect Primer Assay (QIAGEN)• For quantification: QuantiTect SYBR Green PCR Kit (QIAGEN)Appendix 3.1.5: Analysis of entry into G2Cells will be grown in nocodazole-containing medium (20 µg/ml) to arrest them in prometaphase. Fivehours after adding hydrogen peroxide, antioxidant or control, cells will be fixed in 70% ethanol overnight at–20°C. Subsequently, they will be permeabilized with PBS containing 0.1% Triton X-100 for 20 minutes at4 °C, blocked with 2% FBS in PBS, and incubated with 1 µg of anti-pSer10-histone H3 anti-rabbit antibodySCI 332 Advanced Molecular Cell Biology Research Proposal 107