Nitric oxide induces apoptosis in megakaryocytic cell lines

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Nitric oxide induces apoptosis in megakaryocytic cell lines by Elisabeth Battinelli, and Joseph Loscalzo Blood Volume 95(11):3451-3459 June 1, 2000 ©2000 by American Society of Hematology

GSNO and HEL cell endonucleosomal DNA cleavage GSNO and HEL cell endonucleosomal DNA cleavage.(A) Autoradiographic analysis of total cellular DNA prepared from HEL cells after treatment with 100 μmol/L GSNO for different times and 3′-end labeled (1 μg DNA/lane). GSNO and HEL cell endonucleosomal DNA cleavage.(A) Autoradiographic analysis of total cellular DNA prepared from HEL cells after treatment with 100 μmol/L GSNO for different times and 3′-end labeled (1 μg DNA/lane). The characteristic endonucleosomal fragmentation of DNA into 185-bp multiples is visible as early as 30 minutes after treatment with 100 μmol/L GSNO. 1, Untreated HEL cells; 2, HEL cells treated with 100 μmol/L GSNO for 30 minutes; 3, for 1 hour; 4, for 2 hours; 5, for 3 hours; 6, for 4 hours; and 7, for 5 hours. (B) Autoradiographic analysis of total cellular DNA prepared from HEL cells after induction of endogenous iNOS, as described in “Materials and methods.” 1, Cytokine induction of iNOS; 2, cytokine induction of iNOS in the presence of L-NMMA. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

GSNO or iNOS induction and Meg-01 TUNEL assay GSNO or iNOS induction and Meg-01 TUNEL assay.(A) Untreated Meg-01 cells (YOYO-1 DNA stain; 40× magnification). GSNO or iNOS induction and Meg-01 TUNEL assay.(A) Untreated Meg-01 cells (YOYO-1 DNA stain; 40× magnification). (B) Meg-01 cells treated with 100 μmol/L GSNO showing TUNEL-positive cells (YOYO-1 DNA stain; 40× magnification). (C) Percentage apoptotic cells visible by TUNEL assay. To calculate the percentages in each case, 5 to 10 random fields were examined, and each bar graph on the plot represents the average of these experiments (*P < .05). Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

GSNO induction and HEL TUNEL assay GSNO induction and HEL TUNEL assay.(A) HEL cells treated with cytokines to stimulate the expression of iNOS and TUNEL-positive cells (YOYO-1 DNA stain; 40× magnification). GSNO induction and HEL TUNEL assay.(A) HEL cells treated with cytokines to stimulate the expression of iNOS and TUNEL-positive cells (YOYO-1 DNA stain; 40× magnification). (B) HEL cells treated with cytokines to stimulate expression of iNOS and incubated with 100 μmol/L L-NMMA showing decreased TUNEL-positive cell staining (YOYO-1 DNA stain; 40× magnification). (C) Percentage of apoptotic cells that were visible by TUNEL assay. 5 to 10 random fields were examined to calculate the percentages in each case, and each bar in the plot represents the average of 3 experiments (*P < .05). Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

HEL cell CPP32 activity.CPP32 enzymatic activity present in untreated HEL cells, HEL cells treated with a CPP32 inhibitor, and HEL cells treated with 100 μmol/L GSNO. HEL cell CPP32 activity.CPP32 enzymatic activity present in untreated HEL cells, HEL cells treated with a CPP32 inhibitor, and HEL cells treated with 100 μmol/L GSNO. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Meg-01 cell Bcl-2 and Bax expression Meg-01 cell Bcl-2 and Bax expression.(A) Western blot analysis of Bcl-2 expression in Meg-01 cell lysates after treatment with 100 μmol/L GSNO for varying times. Meg-01 cell Bcl-2 and Bax expression.(A) Western blot analysis of Bcl-2 expression in Meg-01 cell lysates after treatment with 100 μmol/L GSNO for varying times. Lane 1, untreated cells; lane 2, 1 hour of treatment; lane 3, 2 hours of treatment; lane 4, 4 hours of treatment. (B) Western blot analysis of Bax expression in Meg-01 cell lysates after treatment with 100 μmol/L GSNO for varying times. Lane 1, untreated; lane 2, 1 hour of treatment; lane 3, 2 hours of treatment. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Peroxynitrite and Meg-01 COMET formation Peroxynitrite and Meg-01 COMET formation.(A) Control Meg-01 cells or (B) Meg-01 cells exposed to 8 μmol/L peroxynitrite for 10 minutes. Peroxynitrite and Meg-01 COMET formation.(A) Control Meg-01 cells or (B) Meg-01 cells exposed to 8 μmol/L peroxynitrite for 10 minutes. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

HEL cells COMET formation and Tiron HEL cells COMET formation and Tiron.Percentage of B and C COMET after treatment of HEL cells with 10 μmol/L GSNO for 120 minutes in the presence of various concentrations of Tiron (circles). HEL cells COMET formation and Tiron.Percentage of B and C COMET after treatment of HEL cells with 10 μmol/L GSNO for 120 minutes in the presence of various concentrations of Tiron (circles). Percentage of B and C COMET after cytokine induction of iNOS in HEL cells for 16 hours in the presence of various concentrations of Tiron (squares). Each point represents the average of 3 experiments. Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Effect of antioxidants on NO-induced HEL cell apoptosis Effect of antioxidants on NO-induced HEL cell apoptosis.Percentage of B and C COMET achieved by exposure to NO produced in co-culture by fibroblasts in which iNOS had been induced in the presence of various superoxide inhibitors, including superoxide dismut... Effect of antioxidants on NO-induced HEL cell apoptosis.Percentage of B and C COMET achieved by exposure to NO produced in co-culture by fibroblasts in which iNOS had been induced in the presence of various superoxide inhibitors, including superoxide dismutase (SOD), catalase, oxypurinol, indomethacin, and Tiron (*P < .02). Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology

Elisabeth Battinelli, and Joseph Loscalzo Blood 2000;95:3451-3459 ©2000 by American Society of Hematology