Cardiolipin (CL) a distinctive mitochondrial phospholipid synthesized by CL synthase (CLS) takes on important yet not fully understood tasks in mitochondria-dependent apoptosis. to apoptosis induced by actinomycin D (ActD) rotenone or gamma-irradiation. During ActD induced apoptosis decreased CL peroxidation along with suppressed cytochrome (cyt) launch were observed in CL-deficient cells while Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely-bound cyt (releasable in high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is definitely catalyzed by CL/cyt complexes and CL oxidation products are essential for cyt launch from mitochondria our results suggest that CL deficiency prevents adequate assembly of effective CL/cyt complexes and CL peroxidation resulting in increased resistance to apoptosis. in CL oxidation . The mechanism includes high affinity relationships of cyt with CL yielding complex with a strong AEB071 peroxidase activity highly specific towards CL. ROS particularly H2O2 are required to feed the peroxidase activity and CL oxidation. Because oxidized CL does not retain cyt  CL oxidation seems to be essential AEB071 for cyt launch from mitochondria [3 5 6 There is general agreement that CL oxidation takes on an essential part in mitochondrial permeabilization and launch of cyt  did not significantly perturb the level and activity of cyt oxidase and overall mitochondrial functions reportedly due to build AEB071 up of the precursor phosphatidylglycerol (PG) that was capable to functionally substitute the requirements in CL . Recently attempts have been made to use the RNA interference (RNAi) protocol to knock-down CLS and decrease CL content in mammalian cells . While the actual level of CL depletion was not accurately assessed based on this approach the authors concluded that inhibition of CLS was associated with an increased level of AEB071 sensitivity to apoptosis – likely due to a weakened binding of cyt AEB071 with CL. In contrast changes of CL induced by mutations of tafazzin gene – whose product is definitely proposed to act as an acyl-transferase influencing the CL redesigning pathway – did not change level of sensitivity to apoptotic difficulties in cells from individuals with Barth syndrome . Therefore the influence of CL content material and/or CL acyl composition on the level of sensitivity of cells to apoptosis is definitely a subject of current medical argument. Characterization of CL’s apoptotic part is definitely important not only for targeted drug discovery but also for understanding of cyt . Materials and methods Materials Rotenone actinomycin D (ActD) antimycin A alamethicin and ATP bioluminescent somatic cell assay kit were purchased from Sigma (St. Louis MO). N-Acetyl-3 7 (Amplex Red) dihydroethidium (DHE) and JC-1 were purchased from Invitrogen (Carlsbad CA). Antibodies against cyt c Bax and Smac/Diablo were from Pharmingen (San Diego CA). Anti COX-IV antibody ENG was from Abcam (Cambridge MA) and anti-actin antibody was from Calbiochem (San Diego CA). HPTLC plates (5 × 5 cm) were purchased from Whatman (Florham Park NJ). All other reagents were purchased from Sigma-Aldrich unless indicated. Generation of stable CLS knockdown HeLa cells with siRNA expression vector Two 19 nucleotide sequences (A: 5’-CCCATGGACAATCCCGAAT-3’; B: 5’-GTGCTCTTGATCCACTTGC-3’) matching human CLS gene (Gene Bank “type”:”entrez-nucleotide” attrs :”text”:”NM_019095″ term_id :”1021087278″ term_text :”NM_019095″NM_019095) were chosen to construct pSEC hygro vector (Ambion Austin TX). In addition a vector containing a siRNA sequence (Ambion) with limited homology to any known sequences in the human genome was used as control. The resulting plasmids were transfected into HeLa cells using Optifect? Reagent (Invitrogen) according to the manufacturer’s instruction. Transfected cells were cultured in the conditioned DMEM medium supplemented with 15% FBS 25 mM HEPES 50 mg/L uridine 110 mg/L pyruvate 2 mM glutamine 1 × nonessential amino acids 2 100 U/ml penicillin and 100 μg/ml streptomycin. The stably transfected clones were selected with 300 μg/ml of hygromycin B. Lipid extraction and 2D-HPTLC analysis Total cellular lipids were extracted as described by Folch  with minor modifications  which allowed highly reproducible quantitative analysis of all lipid fractions including acidic phospholipids. Briefly NaCl was replaced by KCl; the upper phase was.