Dataset Information

Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture protocol

ABSTRACT: Cell line-based proteomics studies are susceptible to intrinsic biological variation that contributes to increasing false positive claims; most of the methods that follow these changes offer a limited understanding of the biological system. We applied a quantitative proteomic strategy (iTRAQ) to detect intrinsic protein variation across SH-SY5Y cell culture replicates. More than 95% of the quantified proteins presented a coefficient of variation (CV) <20% between biological replicates and the variable proteins, which included cytoskeleton, cytoplasmic and housekeeping proteins, are widely reported in proteomic studies. We recommend this approach as an additional quality control before starting any proteomic experiment.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Neuroblastoma

SUBMITTER: JIMMY RODRIGUEZ

LAB HEAD: Magno Junqueira

PROVIDER: PXD010776 | Pride | 2018-08-15

REPOSITORIES: Pride

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Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture protocol.

Murillo Jimmy Rodriguez JR Pla Indira I Goto-Silva Livia L Nogueira Fábio C S FCS Domont Gilberto B GB Perez-Riverol Yasset Y Sánchez Aniel A Junqueira Magno M

Analytical biochemistry 20180823

Cell line-based proteomics studies are susceptible to intrinsic biological variation that contributes to increasing false positive claims; most of the methods that follow these changes offer a limited understanding of the biological system. We applied a quantitative proteomic strategy (iTRAQ) to detect intrinsic protein variation across SH-SY5Y cell culture replicates. More than 95% of the quantified proteins presented a coefficient of variation (CV) < 20% between biological replicates and the v ...[more]

PMID: 30145218

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Project description:Gelsemium sempervirens L. (Gelsemium) is a traditional medicinal plant which at ultra-low doses is employed as anxiolytic and antidepressant, but consistent evidence is lacking and the possible mechanisms involved at the level of nervous system are largely unknown. In this study we have investigated the gene expression of a human neurocyte cell line treated in vitro with increasing dilutions of Gelsemium extract. Starting from a crude extract containing the active principle gelsemine at the concentration of 6.5Ã10â4 M, six centesimal (c) dilutions of Gelsemium were prepared according to the French homeopathic pharmacopoeia: 2c, 3c, 4c, 5c, 9c and 30c. Human neuroblastoma cells SHSY5Y were exposed for 24h to Gelsemium and their transcriptome was compared with control cells treated with the corresponding vehicle solutions. RNA from cells treated with Gelsemium/controls dilutions was isolated and analyzed by microarray. Four biological replicates were tested for each condition. The exposure to the Gelsemium 2c dilution (the highest dose employed, corresponding to 6.5x10-9 M gelsemine) significantly changed the expression of 56 genes, 49 of which were down-regulated and 7 were overexpressed. These genes belong to G-protein coupled protein signalling pathways, calcium homeostasis, inflammatory response and neuropeptide receptors. Cytotoxicity was excluded by WST-1 viability test. The mean fold change of the Gelsemium-downregulated geneset was negative compared to controls in each treatment condition. Although the effect in general decreased with increasing drug dilutions, cluster analysis allowed to identify some groups with typical profile of changes with different dilutions. Among the 49 genes down regulated by Gelsemium 2c, most showed decreased expression after treatment with all dilutions of Gelsemium and comparison with respective controls (Wilcoxon test) was highly significant: 47 genes were down regulated with 3c (p<0.0001), 42 with 4c (p<0.0001), 38 with 5c (p<0.0001), 30 with 9c (p<0.0001), 27 in 30c (p<0.0001). No significant differences of gene expression due to Gelsemium were found in a randomly chosen geneset of the whole cell trascriptome. In conclusion this study showed that Gelsemium, a medicinal plant used in traditional medicine, modulates several genes possibly involved in neuronal function. High throughput microarray technology allowed to detect a small but statistically significant response to extremely low doses/high dilutions, thus revealing an extreme sensitivity of human genome to this kind of pharmacological regulation. A 54 array study that includes both SHSY5Y and IMR32 human neuroblastoma cells samples to investigate the expression changes that result in the treatment with increasing dilutions of Gelsemium sempervirens plant ethanolic extract compared to controls. Each experiment with SHSY5Y cells contained 12 samples. The neurocyte cultures were treated for 24h with 6 dilutions of the Gelsemium sempervirens extract, namely 2c, 3c, 4c, 5c, 9c, 30c (c means centesimal dilution following homeopathic Pharmacopoeia) and the corresponding 6 dilutions of the vehicle solution (controls). Four biological replicates of the experiment were analyzed. IMR32 cells were exposed to Gelsemium 2c dilution and the corresponding control in triplicate experiments.

Dataset Information

Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture protocol

Publications

Mass spectrometry evaluation of a neuroblastoma SH-SY5Y cell culture protocol.

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