Journal: Apoptosis

Article Title: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death

doi: 10.1007/s10495-009-0436-5

Figure Lengend Snippet: Over-expression of neuroglobin reduces sensitivity towards BH3 mimetic-induced cell death. a qRT-PCR ( upper panel , n = 3) and western blot ( lower panel ) analysis of neuroglobin (Ngb) in stable SH-SY5Y-derived cell lines. b Flow cytometry analysis reveals that neuroglobin over-expression partially protects against cell death induced by 2.5 h exposure to 25 μM HA14-1 (mean ± SD, n = 6). Results represent the average from 2 control (pcDNA3.1) cell lines (#2 and #3) and 3 Ngb-over-expressing cell lines (#2, #3, and #4). c Long-term colony survival of wild-type and neuroglobin-over-expressing cells following 48 h treatment with 12.5 μM HA14-1 reveals nearly complete protection by neuroglobin (mean ± SD, n = 10)

Article Snippet: Vector expressing human neuroglobin was generated using a human cDNA clone from Origene (Rockville, MD, USA) subcloned into the KpnI and XbaI sites of pcDNA3.1 and the construct verified by direct sequencing.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Derivative Assay, Flow Cytometry, Expressing

Journal: Apoptosis

Article Title: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death

doi: 10.1007/s10495-009-0436-5

Figure Lengend Snippet: Lowest energy soft-docked structure of the complex between neuroglobin ( left ) and cytochrome c ( right ) calculated using BiGGER and rendered using YASARA ( www.yasara.org )

Article Snippet: Vector expressing human neuroglobin was generated using a human cDNA clone from Origene (Rockville, MD, USA) subcloned into the KpnI and XbaI sites of pcDNA3.1 and the construct verified by direct sequencing.

Techniques:

Journal: Apoptosis

Article Title: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death

doi: 10.1007/s10495-009-0436-5

Figure Lengend Snippet: The activation of apoptosome and caspase-9 depend on neuroglobin concentration. a Neuroglobin = 0, b neuroglobin = 0.01 μM, and c neuroglobin = 0.1 μM. Cytochrome c concentration is uniform at 0.1 μM in all three studies. Time is measured in monte carlo (MC) simulations steps. 1 MC step = 10 −4 s, hence time-scale shown 10 8 MC steps ~2.8 h. Caspase 9 activation is shown (up to 80% completion) normalized by the maximum. Each cross and line represent apoptosome formation and caspase 9 activation, respectively, at a single cell level

Article Snippet: Vector expressing human neuroglobin was generated using a human cDNA clone from Origene (Rockville, MD, USA) subcloned into the KpnI and XbaI sites of pcDNA3.1 and the construct verified by direct sequencing.

Techniques: Activation Assay, Concentration Assay

Journal: Apoptosis

Article Title: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death

doi: 10.1007/s10495-009-0436-5

Figure Lengend Snippet: Probability distribution of caspase 3 calculated from single cell activation data of caspase 3. We show results for two neuroglobin concentrations: neuroglobin = 0 ( white bars ) and neuroglobin = 0.01 μM ( black bars ). Cytochrome c concentration is fixed at 0.1 μM. Caspase 3 concentration ( x -axis) is normalized by the maximum. Time is measured in monte carlo (MC) simulations steps. 1 MC step = 10 −4 s

Article Snippet: Vector expressing human neuroglobin was generated using a human cDNA clone from Origene (Rockville, MD, USA) subcloned into the KpnI and XbaI sites of pcDNA3.1 and the construct verified by direct sequencing.

Techniques: Activation Assay, Concentration Assay

Journal: Apoptosis

Article Title: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death

doi: 10.1007/s10495-009-0436-5

Figure Lengend Snippet: Neuroglobin binding to cytochrome c is sufficient to block activation of caspase 9. a Assay of the apoptosome activation of caspase 9 used the cytosol obtained from cultured Jurkat cells to provide the major components of the apoptosome (dATP, Apaf-1 and pro-caspase 9). Apoptosome activation was initiated by the addition of cytochrome c either in the absence or presence of added neuroglobin, and the subsequent assembly of the apoptosome monitored by following the cleavage of a luminescent peptide substrate for caspase 9. b Caspase 3 activation data without the neuroglobin reduction of cytochrome c to a non-apoptotic form. We used cytochrome c 0.1 μM and neuroglobin 0.1 μM in these simulations. Each line shows the time-course of caspase-3 activation at the single cell level. Caspase 3 activation is normalized by the maximum. Time is measured in monte carlo (MC) simulations steps. 1 MC step = 10 −4 s

Article Snippet: Vector expressing human neuroglobin was generated using a human cDNA clone from Origene (Rockville, MD, USA) subcloned into the KpnI and XbaI sites of pcDNA3.1 and the construct verified by direct sequencing.

Techniques: Binding Assay, Blocking Assay, Activation Assay, Cell Culture

Journal: Apoptosis

Article Title: Neuroglobin protects nerve cells from apoptosis by inhibiting the intrinsic pathway of cell death

doi: 10.1007/s10495-009-0436-5

Figure Lengend Snippet: Increased levels of cytochrome c require higher levels of neuroglobin to block activation of caspase 3. Time course of caspase-3 activation is shown for three different values of cytochrome c concentrations: a 0.2 μM, b 0.5 μM, c 1 μM. Neuroglobin concentration is kept fixed at 0.1 μM. Each line shows the time-course of caspase-3 activation at the single cell level. Caspase 3 activation is normalized by the maximum. Time is measured in monte carlo (MC) simulations steps. 1 MC step = 10 −4 s. Note the variation in time-scales in caspase 3 activation as the cytochrome c concentration is increased

Article Snippet: Vector expressing human neuroglobin was generated using a human cDNA clone from Origene (Rockville, MD, USA) subcloned into the KpnI and XbaI sites of pcDNA3.1 and the construct verified by direct sequencing.

Techniques: Blocking Assay, Activation Assay, Concentration Assay