Cytotoxicity of Superoxide Dismutase 1 in Cultured Cells Is Linked to Zn2+ Chelation
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{"title"=>"Cytotoxicity of Superoxide Dismutase 1 in Cultured Cells Is Linked to Zn2+ Chelation", "type"=>"journal", "authors"=>[{"first_name"=>"Ann-Sofi", "last_name"=>"Johansson"}, {"first_name"=>"Monika", "last_name"=>"Vestling"}, {"first_name"=>"Per", "last_name"=>"Zetterström"}, {"first_name"=>"Lisa", "last_name"=>"Lang"}, {"first_name"=>"Lina", "last_name"=>"Leinartaitė"}, {"first_name"=>"Mikael", "last_name"=>"Karlström"}, {"first_name"=>"Jens", "last_name"=>"Danielsson"}, {"first_name"=>"Stefan L.", "last_name"=>"Marklund"}, {"first_name"=>"Mikael", "last_name"=>"Oliveberg"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"22558346", "sgr"=>"84865856382", "pui"=>"365585774", "scopus"=>"2-s2.0-84865856382", "issn"=>"1932-6203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0036104"}, "id"=>"e27d278e-f777-33d2-99f1-d32e6dc3e0d2", "abstract"=>"Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.", "link"=>"http://www.mendeley.com/research/cytotoxicity-superoxide-dismutase-1-cultured-cells-linked-zn2-chelation", "reader_count"=>25, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>8, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>6, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>4}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>8, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>6, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>4}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>16, "Medicine and Dentistry"=>2, "Chemistry"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>16}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United Kingdom"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/648876"], "description"=>"<p>A set of apoSOD1 mutants with altered protein stability, charge and Zn<sup>2+</sup> binding capacity was added to SH-SY5Y cells in duplicate or triplicate in a concentration range of 0.6 to 10 µM and incubated for 72 h. Cell viability was measured with the MTT assay. (<b>A</b>) Relative cell viability was calculated as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#s4\" target=\"_blank\">methods</a>, and plotted as a function of melting temperature, <i>T</i><sub>m</sub> (Eq.5, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#pone-0036104-t001\" target=\"_blank\">Table 1</a>). A correlation between cell viability and protein stability is observed; overall, the viability is higher for SOD1 mutants with low <i>T</i><sub>m</sub>. SOD1 variants with mutated Zn<sup>2+</sup> ligands as well as [F64A] and [D124G], all with a diminished ability to bind Zn<sup>2+</sup>, fall outside of this pattern (red symbols) (for detailed information, c.f. <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#pone-0036104-t001\" target=\"_blank\">Table 1</a>). The line represents a sigmoidal fit (Eq. 5) of all data points, except those from mutants with diminished ability to bind Zn<sup>2+</sup>. (<b>B</b>) Monomeric apoSOD1 (○) induces a toxic response, whereas [H63/71/80S;D83S] (□), [H71/80S;D83S] (▵), [H71/80S] (▿) and [H71S] (⋄) are all non-toxic. As all of these mutants lack H71, this amino acid was engineered back into [H63/71/80S;D83S] and [H71/80S;D83S], creating the mutants [H63/80S;D83S] (×) and [H80S;D83S] (✶). However, these mutants show no effect on cell viability, indicating that it is the concerted action of the Zn<sup>2+</sup> ligands that underlies the toxicity, not H71 alone. Proteins were added in duplicate and data are presented as mean percentage of buffer control ± range.</p>", "links"=>[], "tags"=>["perturbed"], "article_id"=>319364, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g003", "stats"=>{"downloads"=>1, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ApoSOD1_with_perturbed_Zn_2_site_is_non_toxic_/319364", "title"=>"ApoSOD1 with perturbed Zn<sup>2+</sup> site is non-toxic.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:36:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/649495"], "description"=>"<p>Protein stability (Δ<i>G</i><sub>U/F</sub>) determines the relative concentrations of unfolded ([U]) and folded ([F]) apo protein and, thus, the effective concentration of species able to chelate Zn<sup>2+</sup> from the growth medium. For stable mutations where [F]<sub>apo</sub>≫[U], the protein acts as an efficient Zn<sup>2+</sup> chelator, disturbing the cellular Zn<sup>2+</sup> homeostasis. In the most simplistic case, the apoSOD1 molecules do not interact with the cells but lower the intracellular Zn<sup>2+</sup> levels by preventing reflux from the growth medium.</p>", "links"=>[], "tags"=>["aposod1", "exerts", "toxicity", "cultured"], "article_id"=>319975, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g008", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_outline_of_how_apoSOD1_exerts_toxicity_to_cultured_cells_/319975", "title"=>"Schematic outline of how apoSOD1 exerts toxicity to cultured cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:46:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/648778"], "description"=>"<p>Dimeric and monomeric apoSOD (25 µM) were applied to a Superdex 75 column and the eluted fractions were tested for cytotoxicity in SH-SY5Y cells (black) and analysed for SOD1 by western immunoblotting (red). Cell viability was measured with the resazurin assay and presented as percentage viability of buffer control. The peak areas from the western blot analysis have been normalised to total amount of SOD1 in the chromatography. Cytotoxicity coincides with the dimeric (<b>A</b>) and monomeric (<b>B</b>) peaks of the chromatograms. (<b>C</b>) Analysis of the cell-culture medium directly after protein addition of dimeric apoSOD1 and after 72 h incubation confirms that the apoSOD1 molecules remain dimeric throughout the toxicity assay. Large molecules and aggregates would elute in the void volume of the Superdex-75 column around fraction 26. The concentration of SOD1 in the culture medium was varied between 0.16 µM (black), 0.625 µM (purple), 1.25 µM (red), 2.5 µM (blue) and 12.5 µM (green).</p>", "links"=>[], "tags"=>["aposod1", "molecules", "self-assemble"], "article_id"=>319260, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g002", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_apoSOD1_molecules_do_not_self_assemble_in_the_cell_culture_media_/319260", "title"=>"The apoSOD1 molecules do not self-assemble in the cell culture media.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:34:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/649204"], "description"=>"<p>The high-affinity Zn<sup>2+</sup> chelators TPEN and DTPA were dissolved in DMSO and added to SH-SY5Y cells in parallel with monomeric apoSOD1 in a concentration range of 0.16 to 20 µM, and incubated for 72 h before addition of MTT. The final concentration of DMSO in the cell culture was 0.1%. (<b>A</b>) TPEN (▵) induces a toxic response similar to that of monomeric apoSOD1 (○), whereas DTPA (□) shows an overall weaker effect. Chelators and apoSOD1 protein were added in duplicate and data are presented as mean percentage of vehicle control ± range. (<b>B</b>) Phase contrast images of cells exposed to 10 µM monomeric apoSOD1, 10 µM TPEN and 10 µM DTPA. Cells incubated with apoSOD1 closely resemble cells incubated with TPEN.</p>", "links"=>[], "tags"=>["chelators", "induce"], "article_id"=>319692, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g006", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Zn_2_chelators_induce_a_toxic_response_similar_to_that_of_apoSOD1_/319692", "title"=>"Zn<sup>2+</sup> chelators induce a toxic response similar to that of apoSOD1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:41:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/648989"], "description"=>"<p>The backbone dynamics of monomeric apo- and holoSOD1 evaluated by NMR relaxation experiments. (<b>A</b>) Structural representation of the SOD1 monomer showing the regions of increased dynamic motions in the apo state. (<b>B</b>) Amino-acid sequence of loops IV and VII. (<b>C</b>) Increased dynamics in the intermediate time scale is indicated by increased values of R<sub>1</sub>/R<sub>2</sub> and increased fast dynamics as increased negative values of (NOE-1)R<sub>1</sub>. In the apoSOD1 monomer, the regions of loops IV and VII show increased dynamics <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#pone.0036104-Banci2\" target=\"_blank\">[31]</a>, whereas in the metallated monomer <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#pone.0036104-Banci5\" target=\"_blank\">[71]</a> the loops are as fixed as the rest of the protein. Figure outline adapted from Nordlund <i>et al. </i><a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#pone.0036104-Nordlund1\" target=\"_blank\">[32]</a>.</p>", "links"=>[], "tags"=>["ions", "leads", "backbone", "active-site", "loops", "iv"], "article_id"=>319472, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loss_of_Cu_1_2_and_Zn_2_ions_leads_to_increased_backbone_dynamics_of_the_active_site_loops_IV_and_VII_/319472", "title"=>"Loss of Cu<sup>1+/2+</sup> and Zn<sup>2+</sup> ions leads to increased backbone dynamics of the active-site loops IV and VII.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:37:52"}
  • {"files"=>["https://ndownloader.figshare.com/files/333395", "https://ndownloader.figshare.com/files/333432", "https://ndownloader.figshare.com/files/333499", "https://ndownloader.figshare.com/files/333538", "https://ndownloader.figshare.com/files/333589", "https://ndownloader.figshare.com/files/333647"], "description"=>"<div><p>Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn<sup>2+</sup> ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn<sup>2+</sup> homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn<sup>2+</sup> affinity abolish completely the cytotoxic response. So does the addition of surplus Zn<sup>2+</sup>. Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.</p> </div>", "links"=>[], "tags"=>["cytotoxicity", "superoxide", "dismutase", "cultured", "cells", "linked", "chelation"], "article_id"=>125841, "categories"=>["Neuroscience", "Cell Biology", "Biochemistry"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>[nil, nil, nil, nil, nil, nil], "stats"=>{"downloads"=>8, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Cytotoxicity_of_Superoxide_Dismutase_1_in_Cultured_Cells_Is_Linked_to_Zn_sup_2_sup_Chelation/125841", "title"=>"Cytotoxicity of Superoxide Dismutase 1 in Cultured Cells Is Linked to Zn<sup>2+</sup> Chelation", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-04-25 01:37:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/648669"], "description"=>"<p>Apo and holoSOD1 in dimeric and monomeric form were added to the cell media of cultured SH-SY5Y cells and incubated for 72 h. Proteins were added in duplicate wells and cell viability was measured with the MTT assay. Cell viability is presented as the mean percentage viability of the buffer control ± range. Cell morphology was visualized with immunocytochemistry, using DAPI for DNA staining (blue) and an anti-tubulin antibody for staining of the cytoskeleton (red). (<b>A</b>) Both monomeric (○) and dimeric (▵) apoSOD1 induced a cytotoxic response when added in µM concentrations (calculated as monomeric SOD1 concentration for both dimer and monomer). Neither monomeric (•) nor dimeric (▴) holoSOD1 induce toxicity in the same concentration interval. (<b>B</b>) Monomeric holoSOD1 (5 µM) did not induce any morphological cell alterations after 72 h of incubation (400× magnification). (<b>C</b>) Remaining cells after exposure to 5 µM monomeric apoSOD1 for 72 h displayed an altered morphology characterized by twisted and distorted neurites (400× magnification).</p>", "links"=>[], "tags"=>["viability", "cultured"], "article_id"=>319156, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ApoSOD1_reduces_viability_of_cultured_cells_/319156", "title"=>"ApoSOD1 reduces viability of cultured cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:32:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/649564"], "description"=>"a.<p>The midpoint of the thermal unfolding transition monitored by CD, as derived by fitting of a sigmoidal function (Eq. 3), using a Δ<i>C</i><sub>p</sub> value of 1.4 kcal mol<sup>−1</sup> K<sup>−1</sup>. This value was derived from the slope of Δ<i>H</i><sub>U/F</sub>(T<sub>m</sub>) vs. T<sub>m</sub> for the first 20 mutants in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#pone-0036104-t001\" target=\"_blank\">Table 1</a> (data not shown).</p>b.<p>Mean cell viability expressed as percentage of buffer control with monomeric apoSOD1 [C6/111A;F50E;G51E] set to 0%. Replicate values are displayed in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036104#pone-0036104-g003\" target=\"_blank\">Figure 3A</a>.</p>c.<p>Min/Max represents the minimum and maximum viability response observed for the individual mutations.</p>d.<p>Number of cell viability experiments for the individual SOD1 mutations.</p>", "links"=>[], "tags"=>["viability", "aposod1"], "article_id"=>320049, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.t001", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Melting_temperature_T_m_and_cell_viability_of_apoSOD1_protein_variants_/320049", "title"=>"Melting temperature (<i>T</i><sub>m</sub>) and cell viability of apoSOD1 protein variants.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-04-25 00:00:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/649381"], "description"=>"<p>Monomeric apoSOD1 [C6/111A;F50E;G51E] (<b>1</b>) and the mutants [H46/48/120S] (<b>2</b>), [H71/80S;D83S] (<b>3</b>) and [H46/48/63/71/80/120S;D83S] (<b>4</b>) were added to a final concentration of 10 µM to SH-SY5Y cells loaded with radioactive <sup>65</sup>Zn. Samples of the culture media were collected, concentrated dry on a 10 K filter and exposed to a phospho-imaging screen. As reference for signal quantification, we used lysate of <sup>65</sup>Zn-loaded cells and a standard series of known <sup>65</sup>Zn concentrations. Cell viability was measured in a parallel experiment. (<b>A</b>) Monomeric apoSOD1 [C6/111A;F50E;G51E] and [H46/48/120S], which both show high Zn<sup>2+</sup> affinity, reduce cell viability to below 20%, whereas the low Zn<sup>2+</sup> affinity mutants [H71/80S;D83S] and [H46/48/63/71/80/120S;D83S] have no significant effect on cell viability. (<b>B</b>) Filter radioactivity showing transfer of <sup>65</sup>Zn to the different SOD1 variants in panel A. The signal intensity declines with decreasing Zn<sup>2+</sup> affinity of the apoSOD1 proteins concentrated on the filter. The high-density patterns in 1 and 2 result from manufactured depressions in the filter surface. (<b>C</b>) The molar ratio of Zn<sup>2+</sup> to SOD1 decreases with the Zn<sup>2+</sup> affinity of the protein. 100% is the ratio expected if all <sup>65</sup>Zn present in the cells (quantified to 1.9 fmol/100 000 cells) would transfer to the protein. (<b>D</b>) Plot of cytotoxic response (1-viability %) vs. molar ratio of Zn<sup>2+</sup> to SOD1.</p>", "links"=>[], "tags"=>["transferred", "cytotoxic", "aposod1"], "article_id"=>319868, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g007", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Intracellular_Zn_2_is_transferred_to_cytotoxic_apoSOD1_proteins_/319868", "title"=>"Intracellular Zn<sup>2+</sup> is transferred to cytotoxic apoSOD1 proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:44:28"}
  • {"files"=>["https://ndownloader.figshare.com/files/649098"], "description"=>"<p>10 µM monomeric apoSOD1 was first added to multiple wells of SH-SY5Y cells and then washed away after different times of incubation. In parallel to washing, 10 µM monomeric apoSOD1 was also added to untreated cells. In a second line of experiments, 10 µM monomeric apoSOD1 [H46/48/120S] was added to the cells and, after different times of incubation, 20 µM of ZnCl<sub>2</sub> was added. MTT was added at the endpoint (72 h) of both experiments. Proteins were added in duplicate and data is presented as mean percentage of the buffer control ± range. (<b>A</b>) Washing away the apoSOD1 protein after different length of incubation (○) induces the same toxicity as when adding the protein the same number of hours before the endpoint of the experiment (▪). (<b>B</b>) Adding Zn<sup>2+</sup> to the cell media after different delay times has the same effect on cell viability as washing the protein away. Filled and empty symbols represent separate experiments.</p>", "links"=>[], "tags"=>["toxicity", "builds", "halted"], "article_id"=>319583, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Ann-Sofi Johansson", "Monika Vestling", "Per Zetterström", "Lisa Lang", "Lina Leinartaitė", "Mikael Karlström", "Jens Danielsson", "Stefan L. Marklund", "Mikael Oliveberg"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0036104.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ApoSOD1_toxicity_builds_up_over_time_and_can_be_halted_by_Zn_2_addition_/319583", "title"=>"ApoSOD1 toxicity builds up over time and can be halted by Zn<sup>2+</sup> addition.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-04-25 02:39:43"}

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