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Extended survival of SH-SY5Y cells following overexpression of Lys67Glu neuroglobin is associated with stabilization of ΔψM
Joanna Skommer,
Thomas Brittain,
Corresponding Author
Joanna Skommer
School of Biological Sciences, University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
School of Biological Sciences, University of Auckland, Thomas Bld., 3a Symonds Street, Auckland 1142, New ZealandSearch for more papers by this authorThomas Brittain
School of Biological Sciences, University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
Search for more papers by this authorJoanna Skommer,
Thomas Brittain,
Corresponding Author
Joanna Skommer
School of Biological Sciences, University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
School of Biological Sciences, University of Auckland, Thomas Bld., 3a Symonds Street, Auckland 1142, New ZealandSearch for more papers by this authorThomas Brittain
School of Biological Sciences, University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
Search for more papers by this authorAbstract
Overwhelming evidence indicates that a high level of expression of the protein neuroglobin protects neurons in vitro, in animal models, and in humans, against cell death associated with hypoxic and amyloid insult. We have previously showed that neuroglobin protects neuronal cells from the mitochondrial pathway of apoptosis induced by the BH3 mimetic, by preventing cytochrome c-triggered activation of caspase 9. Here, using cell and molecular biology approaches, we generated a particular neuroglobin mutant, Lys67Glu, overexpression of which confers a significant protection from the BH3 mimetic (TW-37)-induced apoptosis in human neuroblastoma SH-SY5Y cells. The cumulative inhibition of caspase 9 activation is significantly enhanced in Lys67Glu neuroglobin-expressing cells, as compared to wild-type neuroglobin expressing cells. A multiparameter flow cytometry analysis of TW-37-treated cells revealed that inhibition of caspase 9 activity by Lys67Glu neuroglobin is associated with the preservation of the mitochondrial transmembrane potential (ΔψM), as well as a decreased rate of cytochrome crelease from the mitochondria. © 2012 International Society for Advancement of Cytometry
Supporting Information
Additional Supporting Information may be found in the online version of this article.
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| CYTO_22046_sm_SuppFig1.doc53 KB | Figure S1 Expression of EGFP does not affect the pro-apoptotic activity of TW-37 in transiently-transfected SH-SY5Y cells. a) SH-SY5Y cells were transiently transfected with pcDNA_Ngb, in 2 to 1 ratio with either empty pcDNA3.1 or pEGFP, as indicated. 72h post-transfection cells were collected and analyzed by flow cytometry (FL1 channel, FACS Calibur, BD) to assess the transfection efficacy and the level of GFP fluorescence. Plots were generated using FCS Express (De Novo Software, LA, CA, USA). b) SH-SY5Y cells transiently transfected with pEGFP, and 24h later treated with TW-37 for another 48h. At the end of experiment cells were collected, stained with 7-AAD, and analyzed by flow cytometry (channel 1 for EGFP; channel 3 for 7-AAD; FACS Calibur). Cell debris was excluded based on FSC/SSC gating. EGFP+ and EGFP- cells, gated as shown in a), were analyzed based on the FSC/7-AAD plots to determine cell viability. Data are presented as mean±SD (n=3). |
| CYTO_22046_sm_SuppFig2.doc46.5 KB | Figure S2 An increase in the intracellular Ca2+ levels in SY-SY5Y cells following treatment with TW-37 is dampened to a similar extent by the over-expression of Ngb and Lys67Glu Ngb. a) Following treatment of wt SH-SY5Y cells with 10 μM TW-37 for 24h, cells were loaded with cell membrane permeable Fluo-4-AM probe, and basal cytosolic Ca2+ levels determined with the use of flow cytometry (FL1 channel, FACS Calibur). Prior to analysis of Fluo-4 AM fluorescence, cells were gated based on FSC/SSC to exclude dead cells and debris. Plots were generated using FCS Express (De Novo Software, LA, CA, USA). A study representative of 3 independent experiments is shown. b) Quantitative analysis of Fluo-4 fluorescence in wt, Ngb, and Lys67Glu Ngb overexpressing SH-SY5Y cells treated with DMSO or TW-37 (10μM). Prior to analysis of Fluo-4 AM fluorescence, cells were gated based on FSC/SSC to exclude dead cells and debris. Fluorescence levels were baseline subtracted, and then mean ?F was calculated as a difference between mean fluorescence in cells treated with TW-37 and mean fluorescence in cells treated with DMSO. Data are expressed as mean±SEM (n=3). Similar trend was observed when cells were pre-treated with a pan-caspase inhibitor zVAD-fmk (30μM). |
| CYTO_22046_sm_SuppFig3.doc42 KB | Figure S3 Ngb and Lys67Glu neuroglobin do not maintain long-term survival of SH-SY5Y cells pcDNA, Ngb, and Lys67Glu Ngb stable SH-SY5Y cells were treated with DMSO or TW-37 (10μM), continuously for 96h. Next, life and dead cells were collected, stained with 7-AAD and analyzed by flow cytometry (FL3 channel, FACS Calibur). |
| MIFlowCyt.doc42 KB | Supporting Information: MIFlowCyt item location |
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