For H2AX phosphorylation quantitative analysis, foci were counted by eye until at least 40 cells and 40 foci were recorded per sample. Cell extracts and subcellular fractionation Ceritinib FDA Cells were harvested and washed with PBS, pelleted, and lysed in Laemmli buffer containing as inhibitors 1 mM phenylmethylsulfo nyl fluoride, pepstatin, aprotinin, leupeptin and 1 mM sodium orthovana date. Total lysates were boiled for 2 min, sonicated, and quantified by the micro bicinchoninic acid method. Protein content was checked probing the blots with either vinculin or actin specific Abs. For subcellular fractionation, 2 107 T cells were harvested and washed with PBS. mitochondrial and cytosolic fractions were iso lated with the use of the Mitochondria Isolation Kit for cultured cells.

The mitochondrial pellet was lysed in Laemmli buffer, and the cytosolic supernatant was concentrated with a Microcon device. Both fractions were quantified by the micro bicinchoninic acid method before analysis by immunoblotting. Protein content and purity of the fractions were checked probing the blots with vinculin and TOM40 specific Abs. Immunoblot analysis 40 g of total lysates and 20 g of mitochondrial and cytosolic fractions were size fractionated by SDS PAGE 7 to 10% gels and electroblotted onto polyvinylidene diflu oride membranes. After blocking with 5% non fat dried milk in PBS plus 0. 1% Tween, the membranes were incu bated with anti p53 or p53 pSer15, MDM2, BAX, TOM40, p73, p63, vinculin, actin specific Abs and subsequently with peroxidase conjugated secondary antibodies.

The immunoreactive bands were visual ized by ECL Super Signal on autoradi ographic films. Autoradiographic bands were scanned and quantified by Kodak 1D Image Analysis Software. Results The stress response elicited by DRB in human lymphocytes results in DNA replication independent, DNA damage independent and p53 mediated apoptosis We investigated the effects induced by DRB in human cells. To define the sensitivity of cultured T lymphocytes to this drug, we assessed the viability of treated cells in a dose response experiment using propidium iodide staining and flow cytometry. T lymphocytes were suscep tible to DRB induced death within the 40 100 M range in a dose dependent manner. The death mechanism was apoptosis, as both annexin V PI and active caspase 3 cells could readily be detected. Analogous results were obtained with proliferating lymphoblastoid B cell lines and with freshly extracted circulating lym phocytes. All cell lines showed significant sen sitivity to DRB at doses above 40 M, compared to untreated cells. Many Dacomitinib agents that block pol II elongation also block DNA replication.