An Optimized Approach to Recover Secreted Proteins from Fibroblast Conditioned-Media for Secretomic Analysis

Paré, Bastien; Deschênes, Lydia Touzel; Pouliot, Roxane; Dupré, Nicolas; Gros‐Louis, François

doi:10.3389/fncel.2016.00070

Cited by 19 publications

(15 citation statements)

References 55 publications

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“…Total cell soluble proteins were prepared as described previously [22]. The trichloroacetic acid (TCA)-deoxycholate (DOC) protein precipitation method was used to concentrate proteins in cell culture-conditioned media as previously reported [23]. Briefly, three 10-cm plates of adherent cells were cultured in DMEM with FBS until confluence, when the medium was replaced with fresh medium without FBS for an additional 1 day.…”

Section: Protein Sample Preparationmentioning

confidence: 99%

“…Red-colored genes were selected for further qPCR-based gene expression confirmation. Bar graphs of selected a neurotrophic factors, b neuroprotective factors, and c growth factors genes are present in the cell culture media, we harvested 2day media of confluent cultures and concentrated the proteins in the media using the trichloroacetic acid (TCA) precipitation protocol [23]. The concentrated protein samples were thereafter electrophoresed by SDS-PAGE for WB analysis [22].…”

Section: Detection Of Factors That Cells Secreted Into the Cell Cultumentioning

confidence: 99%

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Paracrine effects of intraocularly implanted cells on degenerating retinas in mice

Liu

Chen

et al. 2020

Stem Cell Res Ther

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Background: Retinal degeneration is a leading cause of blindness in the world; its etiology is complex and involves genetic defects and stress-associated aging. In addition to gene therapies for known genetically defective retinal degeneration, cellular therapies have been widely explored for restoring vision in both preclinical animal models and clinical trials. Stem cells of distinct tissue sources and their derived lineages have been tested for treating retinal degeneration; most of them were reported to be effective to some extent in restoring/improving deteriorated vision. Whether this visual improvement is due to a functional integration of grafted cells to substitute for lost retinal neurons in recipients or due to their neuroprotective and neurotrophic effects to retain recipient functional neurons, or both, is still under debate. Methods: We compared the results of subretinal transplantation of various somatic cell types, such as stem cells and differentiated cells, into Rho P23H/+ mice, a retinal degeneration model for human retinitis pigmentosa (RP) by evaluating their optokinetic response (OKR) and retinal histology. We identified some paracrine factors in the media that cultured cells secreted by western blotting (WB) and functionally evaluated the vascular endothelial growth factor Vegfa for its potential neurotrophic and neuroprotective effects on the neuroretina of model animals by intravitreal injection of VEGF antibody. Results: We found that live cells, regardless of whether they were stem cells or differentiated cell types, had a positive effect on improving degenerating retinas after subretinal transplantation; the efficacy depended on their survival duration in the host tissue. A few paracrine factors were identified in cell culture media; Vegfa was the most relevant neurotrophic and neuroprotective factor identified by our experiments to extend neuron survival duration in vivo. Conclusions: Cellular therapy-produced benefits for remediating retinal degeneration are mostly, if not completely, due to a paracrine effect of implanted cells on the remaining host retinal neurons.

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Section: Protein Sample Preparationmentioning

confidence: 99%

Section: Detection Of Factors That Cells Secreted Into the Cell Cultumentioning

confidence: 99%

Paracrine effects of intraocularly implanted cells on degenerating retinas in mice

Liu

Chen

et al. 2020

Stem Cell Res Ther

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“…The TCA precipitation method was performed to analyze secreted proteins in the conditioned medium as previously described [ 35 ]. 0.5–1 mL conditioned medium was collected and trichloroacetic acid (Macklin, China) was added to a final concentration of 10%, then vortexed for 1min and incubated on ice for 24 h. The samples were centrifuged at 13000 rpm for 20 min at 4 °C, and the supernatant was discarded and washed twice using 100% ice-cold acetone.…”

Section: Methodsmentioning

confidence: 99%

Adipocyte-Derived Leptin Promotes PAI-1-Mediated Breast Cancer Metastasis in a STAT3/miR-34a Dependent Manner

Wei

Zhan

et al. 2020

Cancers

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The crosstalk between cancer cells and adipocytes is critical for breast cancer progression. However, the molecular mechanisms underlying these interactions have not been fully characterized. In the present study, plasminogen activator inhibitor-1 (PAI-1) was found to be a critical effector of the metastatic behavior of breast cancer cells upon adipocyte coculture. Loss-of-function studies indicated that silencing PAI-1 suppressed cancer cell migration. Furthermore, we found that PAI-1 was closely related to the epithelial-mesenchymal transition (EMT) process in breast cancer patients. A loss-of-function study and a mammary orthotopic implantation metastasis model showed that PAI-1 promoted breast cancer metastasis by affecting the EMT process. In addition, we revealed that leptin/OBR mediated the regulation of PAI-1 through the interactions between adipocytes and breast cancer cells. Mechanistically, we elucidated that leptin/OBR further activated STAT3 to promote PAI-1 expression via miR-34a–dependent and miR-34a–independent mechanisms in breast cancer cells. In conclusion, our study suggests that targeting PAI-1 and interfering with its upstream regulators may benefit breast cancer patients.

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“…Additionally, acetone may also act as a desalting agent [52]. Therefore, the addition of this solvent to the bottom phase can also contribute to remove some phase-forming salts by coprecipitation [53].…”

Section: Partial Purification Of Esterasementioning

confidence: 99%

Integrated strategy for purification of esterase from Aureobasidium pullulans

Lemes

Silvério

Rodrigues

et al. 2019

Separation and Purification Technology

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Esterases catalyze the cleavage and formation of ester bonds of a broad range of substrates presenting a widespread spectrum of industrial applications. This work aimed to partially purify and characterize an esterase from Aureobasidium pullulans LABIOTEC 01 produced in a culture medium containing olive mill wastewater. Esterase purification was evaluated using different strategies, namely the enzyme recovery by PEG-salt aqueous twophase systems (ATPSs); and the enzyme precipitation with ammonium sulfate, acetone, and ethanol. The best purification factor (18 ± 2) was obtained when the ATPS composed of 20% (w/w) polyethylene glycol (PEG) 6000 and 5.8% (w/w) potassium phosphate buffer (PPB) pH 8.0 was combined with acetone precipitation. The partially purified enzyme presented an optimum pH of 5.0, although it remained active in the pH range of 4.5 to 7.5 (≥ 50% relative activity). The optimum temperature was found to be 60°C. Furthermore, the addition of salts such as FeCl 3 , CuSO 4 and MnCl 2 promoted an increase in the enzymatic activity (above 100%). The enzyme was found to be stable and showed high activity when exposed to polar solvents such as dimethyl sulfoxide, dimethylformamide, and methanol. The use of an integrated strategy of purification combining simple purification methods such as ATPS and precipitation was herein reported for the first time for esterase. This strategy proved to be an interesting approach to partially purify the esterase produced under submerged fermentation by A. pullulans. Furthermore, the enzyme showed potential to be applied in industrial biocatalytic processes using high temperature and different salts or solvents. Also, the production of esterase using olive mill wastewater as substrate demonstrated to be a suitable alternative to reduce and valorize agro-industrial residues.

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An Optimized Approach to Recover Secreted Proteins from Fibroblast Conditioned-Media for Secretomic Analysis

Cited by 19 publications

References 55 publications

Paracrine effects of intraocularly implanted cells on degenerating retinas in mice

Paracrine effects of intraocularly implanted cells on degenerating retinas in mice

Adipocyte-Derived Leptin Promotes PAI-1-Mediated Breast Cancer Metastasis in a STAT3/miR-34a Dependent Manner

Integrated strategy for purification of esterase from Aureobasidium pullulans

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