PONE-D-22-09312Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cellsPLOS ONE

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Reviewers' comments:

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Comments to the Author

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study by Fernández-Gallardo et al. explore the expression of α2δ1-protein and related proteins in glioblastoma cells to elucidate the impact of α2δ1 in cell proliferation. Based on indications in the literature the authors explored to which extend α2δ1 expression correlates with the proliferation of U87 cells. Here they used pharmacological antagonists and siRNA mediated modulation of α2δ1 expression to explore the impact of the α2δ1 protein on cell proliferation and intracellular Nfkb mediated signalling cascades. The use of the glioma cell line provides a good model for a channel independent exploration of the α2δ1 mediated aspects in cell proliferation/migration apart from the classical function as calcium channel accessory subunit.

The data suggest a direct action of α2δ1 protein expression as response to LPS stimulation. To further narrow the specificity of LPS induced proliferation a positive control via the application of Thrombospondin-1 would strengthen the correlation between the stimulus and the impact of α2δ1-proteins as receptor molecule of Throbospondin-1. Since Thrombospondins are also for neurons relevant ECM molecules that influence synaptogenesis, such experiment could hint to specific possible interactions between glioma cells and their cellular environment.

Within the western blot analysis of expression, the data have been normalized to the control, but the controls missing an indication of the standard error. To better interpret the significance of the effects such information would be very helpful to see how reliable/stable are the western blot experiments. Please include such error information into the graphs.

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Reviewer #1: No

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Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cells

PONE-D-22-09312R1

Dear Dr. Felix,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Alexander G. Obukhov, Ph.D.

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper focus on the function of a2d1-subunit as a proliferation stimuli within U87 human gliablastoma cells. Following several hypothetical molecular pathways the authors show a connection between the toll-like receptor TLR-4, the transcription factor Sp1 and a2d1, as one of the target genes for this transcription factor. The proliferation effect was measured and modulated either by overexpression or downregulation of interacting proteins or pharmacological treatments. Here gabapentin and thrombospondin were used to block or stimulate a2d1-subunits, respectively. The essays used are robust and all experiments have been done with the needed controls.

The study show additional channel independent function of a2d-proteins, as seen also in neurons and impose the question about the mechanism of this channel independent signalling, which hopefully is subject of further studies.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

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