PONE-D-21-18235Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell culturesPLOS ONE

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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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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?

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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4. 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

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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: In the manuscript by Hall et al., together with their previous paper (ref. 18-20) the authors generated Mgat1 (encodes GnT-I) knock out neuroblastoma cells by CRIPR/Cas9 and obtained mutant cells with aberrant N-glycosylation. The mutant cells showed altered cell morphology and growth properties compared to wild-type parental cells. The authors also showed that the expression of several proteins including MMP, receptor-tyrosine kinase and cytoskeletal proteins were significantly changed in mutant cells.

The experiment seems to be well designed and conducted, and the results ae convincing to support their conclusion. The manuscript is well constructed and written in precise English.

Even though the manuscript largely lacks the mechanistic insights how N-glycans regulate and what proteins is most responsible for these cellular phenotypes, the phenomena itself might be informative to the reader of glycobiology and tumor biology.

I think the publication of the manuscript can be considered if the following concerns are addressed.

Major point

1. through the manuscript

GnT-1 and GnT-2 should be GnT-I and GnT-II (Roman, not Arabian)

2. The schematic drawing to show N-glycans structures and how GnT-I and GnT-II work on the structures should be briefly presented on Figure 1A for beginner readers.

3. Figure 1D

The samples should be run on the same gel and blotted on the same membrane for each blot.

4. Figure 9A

The image is very poor and the samples should be run on the same gel and blotted on the same membrane for each blot.

5. The authors should more discuss the relevance of their finding to the human clinical situations. For instances, human neuroblastoma is classified into stage 1 to 4 and 4S. Do you have any information about the expression of GnT-I with different stages?

Minor point

Lime 108

paladin>paladin

Line 132

two dimensional (2D) versus three dimensional (3D) > 2D and 3D

the abbreviations were already defined and not needed here

Figure 1C

“NB_1 (Mgat1)” should be “NB_1 (-Mgat1)”

Figure 2A

the scale bar should be included into the image

Figure 2B

What is OG? Outgrowth? “Neurite Length” and “Neurite Width” are better.

Figure 2D

Need the scale bar.

Figure 3B

Need the scale bar.

Figure 3C Y-axis

What is the unit?

Figure 3D

Need the scale bar.

Figure 5A and 5B

Need the scale bar

Figure 5C, Y-axis

Should be “Cell invasion index (ratio to spheroid size)”

Figure 5D, Y-axis

Need the unit pixels?

Figure 6

See the comment on Figure 5.

Figure 7B and 7D, Y-axis

Should be “Gelatinase activity (Mutant/NB_1)

Figure 8A

The cell line names are missing.

Figure 9C and 9D, Y-axis

Should be “Band intensity (Mutant/NB_1)” or “Relative expression (Mutant/NB_1)”.

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Reviewer #1: Yes: Kazuma Sakamoto

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Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures

PONE-D-21-18235R1

Dear Dr. Schwalbe,

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,

Salvatore V Pizzo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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: All comments have been addressed

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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

**********

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

**********

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 authors well addressed my concerns.

I do not have additional requirements and recommend the publication of the manuscript in PLOS One.

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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: Yes: Kazuma Sakamoto