Dear Dr. Nashimoto,

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Hodaka Fujii, M.D., Ph.D.

Academic Editor

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?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: No

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

Reviewer #1: The authors use 14-nt guide RNAs to recruit tRNase ZL to the MALAT1 noncoding RNA, resulting in cleavage and knockdown. 6 guides are tested, with some successfully knocking down MALAT1 to varying degrees. The authors confirm these effects require tRNase ZL. The manuscript is overall straightforward but I do have several suggestions.

(1) Introduction, First paragraph: It would be helpful to include a few additional sentences about the TRUE technology and the requirements for oligo design. This will allow a reader to better understand the technology without having to refer to other previous papers.

(2) Fig 3: Only one replicate has been shown. These experiments should be repeated 3 times to show reproducibility.

(3) Fig 5: The authors should comment on why the degree of knockdown obtained in Fig 5B is much stronger than that in 5C,D.

Minor point:

(4) MALAT1 is only 7 kb in length, not 10.5 kb as the authors claim on p.9.

Reviewer #2: The manuscript designed a 14-nt sgRNA compatible with tRNase ZL for cleavage of MALAT1 RNA, which validated the potential of TRUE silencing technology in targeting long non-coding RNAs. The authors determined the efficiency of cleavage by in vitro assay, subcellular localization and gene knock down. However, there remains several concerns:

1. The authors mentioned the nuclear localization of MALAT1 RNA in A549 cell line. However, they didn’t give any experimental evidence or literature citations.

2. Introduction section. It is mentioned that ASO has been used to KD MALAT1. Please discuss the comparison between ASO and TRUE for MALAT1 RNA knock down.

3. The authors should discuss why sgRM3 (sequence worked in ASO) was invalid in TRUE silencing.

4. Fig 3. Negative controls should be considered (non-target sgRNA, or sgRM3,4,5).

5. Where does the sequence of “5’-Alexa568 labeled sgRNA” come from? Why did the authors choose this one for tracing but not the sequence of sgRM1~6, which seems more direct? Will sgRM1~6 exhibit same localizations with this one?

6. Fig 4. Add more controls to exclude the possibility of non-specific signals.

7. Fig 5 legend. Does the term “untreated cells” mean the mock control? If so, please maintain the consistent address throughout the text. (write “mock control” directly)

8. Fig 5B~D. It’s still possible that the phenomenon was caused by indirect effect of impaired tRNA processing. Exclude this possibility experimentally (for example, transfect scrambled sgRM sequence as negative control).

9. Introduction section. The sgRNAs contain three modifications (as described in Materials and Methods), but the authors just mentioned two here in Introduction (lost “six phosphorylation modifications at the 5' and 3' ends”). Please supplement this information to avoid any misunderstanding by readers.

10. In the Materials and Methods section, "and a 5’-Alexa568-labeled, 3’-phosphorylated, fully 2’-O-methylated sgRNA with full phosphorothioate linkages (5’-UUGGUACCUUCUCA-3’)" lacks a name and description of its purpose, making it difficult to understand. It should be given an abbreviation like other sgRNAs.

11. Please add full names of abbreviations such as “FAM” when they are first mentioned.

12. "The targets TM1 and TM2 were cleaved by the corresponding sgRNA-guided tRNase ZL at the expected sites, although the exact cleavage sites were not determined (Fig 3A, B). Likewise, the target TM6 was cleaved by sgRM6-guided tRNase ZL (Fig 3C)." It’s confusing. To my understanding, the exact cleavage sites of TM1, TM2, TM6 all were not determined, please re-write it more clearly.

13. The term "TRUE gene silencing" is introduced without contextualizing it against existing RNA-targeting technologies (e.g., CRISPR-Cas13, ASOs). A brief comparison is needed to clarify its novelty, mechanistic distinctions, and the deletion effiency.

14. A scrambled/non-targeting sgRNA control is absent in experiments targeting MALAT1 RNA (a “mock” control is insufficient). Its inclusion is essential to confirm sequence-specific cleavage. Moreover, the authors directly transfected sgRNA but did not assess transfection efficiency (e.g., cellular uptake efficiency, sgRNA half-life in cells/serum), limiting mechanistic interpretation.

15. Only one primer pair used for qRT-PCR to quantify MALAT1 (>10,000 nt), risking biased results. The authors should design primer pairs spanning multiple regions (e.g., 5′, mid, and 3′) that are necessary to confirm transcript degradation. Moreover, RNA-seq should be performed to rule out transcriptome-wide unintended effects.

16. Fig. 2A shows only two biological replicates (left panels), and lacks error bars and statistical tests. The authors should expand to n ≥ 3 biological replicates and ensure all figure legends explicitly describe error bars and statistical methods.

17. In Fig. 3, the polyacrylamide gel shows only one short band after cleavage of the 30-nt RNA product. The authors should explain why two expected short cleavage bands are not observed. And the in vitro cleavage assay lacks a non-targeting/scrambled sgRNA control to confirm specificity.

18. While fluorescence microscopy suggests nuclear localization of sgRNA and tRNase ZL in Fig. 4, no significant co-localization is evident in the images. The authors should provide quantitative metrics (e.g., Pearson’s correlation coefficient) to substantiate spatial association.

19. Protein size markers are missing in Figure 5A (western blot). And panels B–D lack non-targeting/scrambled sgRNA controls, error bars, and statistical tests. “Rescue” experiments are required to strengthen conclusions.

20. Figure 6 lacks statistical tests and direct evidence for apoptosis pathways (e.g., caspase activation assays). The authors should perform caspase activity assays or related experiments to validate cell apoptosis.

21. There are lots of un-standard expressions throughout the manuscripts. Please check it thoroughly. Here are some examples:

- Fig 1 legend. Change "An arrow represents an expected cleavage site" to "The expected cleavage sites of target/sgRNA pairs are indicated by arrows."

- Last sentence of Materials and Methods: Change "After 96-hr culture, cell viable was measured using Cell Counting Kit-8" to "After 96 hours of culture, cell viability was measured using the Cell Counting Kit-8."

- Original sentence: "the MALAT1 RNA in the cells was quantitated by qRT-PCR." Suggested revision: "the MALAT1 RNA levels in the cells were quantified by qRT-PCR."

- “While all the sgRNAs showed little effect on the MALAT1 RNA level in 24 and 48 hr”. the word "in" should be replaced with "at" in this sentence, as well as in several other instances throughout the text.

Reviewer #3: Comments/suggestions to the authors:

Introduction

Authors should provide a more precise introduction, detailing the potential benefits of their testing gene silencing approach in comparison to the well-established RNAseH-based method.

Additionally, there is no clear rationale for choosing the 14-nt linear type of sgRNA design among the four available options.

The introduction should be rewritten to enhance clarity.

Results

The specific properties such as Tm, GC%, and potential secondary structure of the selected sgRNAs should be presented in a table or other format to facilitate the comparison of sequences.

The entire package of results is variable and lacks consistency, raising questions about the validity of the effect. In experiments like this, it is critical to have appropriate controls, which are missing in this piece of work. Single-stranded oligonucleotides at a high concentration similar to used in this manuscript can have many indirect effects. Therefore, mock controls are not sufficient; a scramble control should be used instead. This is crucial for all data presented in this manuscript.

Fig. 3 gels potentially can be stained with intercalating dye instead of FAM, allowing for control lanes with sgRNA or target sequence only. The data should include the same time points with and without the enzyme to confirm that degradation is really enzyme-dependent!

While the in vitro cleavage assay has limitations, the authors should provide 5'/3 ‘RACE assay results to demonstrate that the introduced sgRNA cleaves the target in cells.

The paper would be enhanced by demonstrating Malat1 KD effects in other cell types and proving data that this KD approach works on other targets.

Cell viability data is missing the appropriate negative control and also a positive control, which could be scramble ASO and a gapmer targeting the Malat1 transcript, respectively.

Reviewer #4: The manuscript by Takahashi and Nashimoto reports on re-examining a post-transcriptional silencing system that the last author attempts to develop ever since 1991. This system (TRUE) is based on exploiting a ubiquitous endogenous tRNA processing enzyme called RNase Z and exogenous small RNAs to target non-tRNAs. While the system has been used by the Nashimoto et al., to target blood cancers, it seems not to have been widely accepted nor been taken up for further development by others.

In this manuscript, the authors use the system to target a ‘jackpot’ long non-coding RNA called MALAT1, which is implied in pretty much everything, including to function in a variety of cancers. Using a stabilized small RNA to target RNase Z to MALAT1, the authors show that MALAT1 RNA can be reduced, which results in lower cell viability of one lung cancer-derived cell line.

The manuscript is rather simple in its message, and does not really add understanding to the mechanism or improvement of the TRUE system. Overall, the presentation of the few data points are acceptable for publication in PLoS One. However, some obvious questions remain to be answered before publication can be recommended. This reviewer therefore asks for a major revision.

General comments:

(1) While the early attempts to use RNase Z to downregulate the function of non-tRNAs predated the rise of RNAi- and miRNA-based gene regulatory systems, it is curious why the authors are still trying to improve TRUE rather than accepting that other RNA-based approaches are much more developed and more efficient than TRUE. Could the authors give the reader a rationale for using TRUE, and not siRNA-based methods when trying to interfere with MALAT1 function?

(2) 200 nM sgRNA with a size of 14 nt corresponds to 900.9 micrograms of sgRNA per liter.

For transfections in a 12-well format with 1 mL medium this would amount to 900.9 nanograms per well. A 12-well dish format should be seeded at 0.1 x 10^6 cells. Assuming transfection about 24 hours later, after at least one cell division of A549 cells, 0.2 x 10^6 cells will be transfected with 900.9 nanograms. This mass corresponds to 1.204e+14 sgRNAs. Even with a transfection efficiency of only 1%, each cell would be exposed to 6.02e+6 sgRNA molecules. Given the efficiency of MALAT1 downregulation by a maximum of 58%, one wonders if the effects of the knockdown (cell viability reduction) are really due to the loss of function of half the MALAT1 molecules, or due to poisoning of the cells by exorbitant numbers of small RNAs. This aspect of quantitative mass effects should be discussed by the authors.

(3) Given that the authors used qRT-PCR to determine the relative levels of MALAT1 RNA after sgRNA transfection, they should provide the reader with a description where exactly in the MALAT1 RNA sequence the sgRNAs are targeting and where the qRT-PCR primers would bind to the cDNA derived from MALAT1. If the PCR primers were designed to bind to cDNA regions that do not span the RNase Z cleavage site, the reader could assume/conclude that the reported knock-down efficiencies are masked by remnants of MALAT1 RNA that can still be reverse-transcribed.

(4) The authors did not adhere to the PLOS Data policy. “For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available.”

No data points have been submitted with the manuscript.

Specific comments:

(1) “In order to demonstrate that the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level is caused by TRUE gene silencing, we performed in vitro tRNase…”

> It is unclear why the authors resort to an in vitro cleavage experiments as proxy to show that 14 nt sgRNA targets MALAT1. Even though MALAT1 is 6.98 kb transcript, it has been detected by northern blotting, and it is necessary to perform northern blotting with probes around the sgRNA target sides to show that TRUE is targeting MALAT1 after transfection, and to complement the qRT-PCR results. Only then, can the authors claim that MALAT1 is affected by RNase Z-mediated cleavage.

(2) Figure 2A:

> Can the authors explain why transfection of some sgRNAs directed against MALAT1 increase relative MALAT1 transcript levels?

(3) Figure 3:

> To make such an experiment more scientifically sound, could the authors incorporate more cleavage efficiencies that would add to the quantification shown to the right of each PAGE gel? As of now, it looks like this experiment was performed only once, which is not sufficient to accommodate to the scope of PLoS One, a journal that aims to publish experimentally sound manuscripts.

(4) Figure 5:

“(B−D) A MALAT1 RNA amount was measured by qRT-PCR, normalized against a β-actin mRNA amount, and expressed as a percentage relative to that of untreated cells. Data were from three biological replicates.”

> Even though the authors state that they performed biological replicates, the Figure does not show that. Was every replicate output exactly the same?

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

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Dear Dr. Nashimoto,

Please submit your revised manuscript by Aug 23 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #4: Yes

**********

Reviewer #1: The authors have appropriately revised the manuscript and addressed the prior comments from each of the reviewers.

Reviewer #2: The authors have addressed almost all my concerns, although the RNA-seq experiments have not been performed.

Reviewer #4: The authors have addressed some but not all of the comments by this reviewer. However, the authors performed some experiments that can be accepted as substitutes for the requested approaches. This reviewer recommends publication after minor revision.

General comments:

(1) While the early attempts to use RNase Z to downregulate the function of non-tRNAs predated the rise of RNAi- and miRNA-based gene regulatory systems, it is curious why the authors are still trying to improve TRUE rather than accepting that other RNA-based approaches are much more developed and more efficient than TRUE. Could the authors give the reader a rationale for using TRUE, and not siRNA-based methods when trying to interfere with MALAT1 function?

>> The authors have added the following statement:

“Under such circumstances, one may think that clinical applications of TRUE gene silencing would not be worth pursuing. However, we believe that we would have a chance to find a niche of this technology, since there would be a large number of potential target RNAs that cause various diseases and a subset of them may not be able to be eliminated easily by the above established and emerging technologies.”

>> The reviewer understands that the authors are mixing the world of belief with the world of science, the latter of which should be disconnected from belief as much as possible when performing experiments to understand the natural world.

(2) 200 nM sgRNA with a size of 14 nt corresponds to 900.9 micrograms of sgRNA per liter.

For transfections in a 12-well format with 1 mL medium this would amount to 900.9 nanograms per well. A 12-well dish format should be seeded at 0.1 x 10^6 cells. Assuming transfection about 24 hours later, after at least one cell division of A549 cells, 0.2 x 10^6 cells will be transfected with 900.9 nanograms. This mass corresponds to 1.204e+14 sgRNAs. Even with a transfection efficiency of only 1%, each cell would be exposed to 6.02e+6 sgRNA molecules. Given the efficiency of MALAT1 downregulation by a maximum of 58%, one wonders if the effects of the knockdown (cell viability reduction) are really due to the loss of function of half the MALAT1 molecules, or due to poisoning of the cells by exorbitant numbers of small RNAs. This aspect of quantitative mass effects should be discussed by the authors.

>> The authors have added a Figure that shows that exorbitant amounts of non-targeting RNAs increase rather than decrease cell viability. While this reviewer will accept this experiment, the results are contrasting years of research into the poisonous effects of large copy numbers of siRNAs when introduced into human cells.

(3) Given that the authors used qRT-PCR to determine the relative levels of MALAT1 RNA after sgRNA transfection, they should provide the reader with a description where exactly in the MALAT1 RNA sequence the sgRNAs are targeting and where the qRT-PCR primers would bind to the cDNA derived from MALAT1. If the PCR primers were designed to bind to cDNA regions that do not span the RNase Z cleavage site, the reader could assume/conclude that the reported knock-down efficiencies are masked by remnants of MALAT1 RNA that can still be reverse-transcribed.

>> The authors have addressed this point to completion.

(4) The authors did not adhere to the PLOS Data policy. “For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available.”

No data points have been submitted with the manuscript.

>> The authors have addressed this point to completion.

Specific comments:

(1) “In order to demonstrate that the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level is caused by TRUE gene silencing, we performed in vitro tRNase…”

> It is unclear why the authors resort to an in vitro cleavage experiments as proxy to show that 14 nt sgRNA targets MALAT1. Even though MALAT1 is 6.98 kb transcript, it has been detected by northern blotting, and it is necessary to perform northern blotting with probes around the sgRNA target sides to show that TRUE is targeting MALAT1 after transfection, and to complement the qRT-PCR results. Only then, can the authors claim that MALAT1 is affected by RNase Z-mediated cleavage.

>> The authors have addressed this point by performing some complicated 3’ RACE experiment. They did not perform a much simpler northern blot. The results presented in the new Figures are summarized in a complicated statement: “On the whole, the 3′-RACE data would not be inconsistent with the supposition that the 14-nt sgRNAs can guide tRNase ZL to cleave the MALAT1 RNA also in the cells.” This reviewer leaves the conclusion about the in vivo cleavage efficiency to the future readers of this manuscript.

(2) Figure 2A:

> Can the authors explain why transfection of some sgRNAs directed against MALAT1 increase relative MALAT1 transcript levels?

>> The authors give a handwaving explanation that does not really address the discrepancy in perception of these results. As it stands, sgRNAs should result in the degradation of MALAT1 by RNaseZ, but the authors suggest that some sgRNAs result in “slower degradation by disturbed nuclease activity that is caused by sgRNA binding.” This reviewer wonders about the applicability of the method if one has to screen candidate sgRNAs not only for knock-down efficiency of the target RNA, but also for potential stabilization of the target RNA, which is not what the method should achieve.

(3) Figure 3:

> To make such an experiment more scientifically sound, could the authors incorporate more cleavage efficiencies that would add to the quantification shown to the right of each PAGE gel? As of now, it looks like this experiment was performed only once, which is not sufficient to accommodate to the scope of PLoS One, a journal that aims to publish experimentally sound manuscripts.

>> The authors have addressed this point to completion.

(4) Figure 5:

“(B−D) A MALAT1 RNA amount was measured by qRT-PCR, normalized against a β-actin mRNA amount, and expressed as a percentage relative to that of untreated cells. Data were from three biological replicates.”

> Even though the authors state that they performed biological replicates, the Figure does not show that. Was every replicate output exactly the same?

>> This reviewer accepts this explanation.

NEW COMMENTS:

SFig 9:

This Figure is called in the text as: “The sgRNA was observed to be distributed ubiquitously in A549 cells 48 hr after transfection, with higher density in the nucleus (Fig 4 and S9 Fig.)”.

On the other hand, the Figure legend to SFig. 9 reads:

“Fluorescence microscope image of mock-transfected A549 cells. As a control experiment, A549 cells were mock-transfected. After 48-hr culture, the cells were incubated with an Alexa488-conjugated secondary antibody without incubating with primary antibodies against a human tRNase ZL peptide, and analyzed with a fluorescence microscope. Hoechst 33342 was used to stain the nucleus. Each image was taken by irradiating a laser beam of the indicated wave length.”

>>To this reviewer there is some discrepancy between the two statements, especially since SFig. 9 does not show only the nuclear stain and no signal in the other two emission channels.

**********

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

Reviewer #2: No

Reviewer #4: No

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

Cleavage of MALAT1 RNA by 14-nt sgRNA-guided tRNase ZL

PONE-D-25-04390R2

Dear Dr. Nashimoto,

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,

Hodaka Fujii, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

**********

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

Reviewer #4: Yes

**********

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

Reviewer #4: Yes

**********

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

The PLOS Data policy

Reviewer #4: Yes

**********

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

Reviewer #4: Yes

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Reviewer #4: The authors clarified and answered the remaining question by this reviewer concerning the call of Fig.4 and SFig.9 in text.

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Reviewer #4: No

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