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Your revision is now acceptable for publication and we appreciate you for the careful address and correction.
The present manuscript has some new informationson the novel roles of poly-ADP-ribose (PAR) in the cytoskeletal potentials. The logic explanation how TNKS-1 displays for the PAR function in the cytoskeletal arrangements should be discussed in the appropriate section during your revision. We understand this study is not the final destination of the TNKS-1-PAR-cytoskeletal collaborative expression.
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In the paper, the authors suggest the existence of PAR in a novel subcellular localization.
The paper may be acceptable for publication provided major corrections are made and additional information is given. Please refer to the details below.
Major issues:
1-How is PARP and TNKS-1 expressed in VERO cells? The authors only show PAR accumulation, but they must also show colocalisation of PAR-TNKS-1, and PAR-PARP1 on VERA cells. And the authors must confirm the result with western blot analysis for PARylated actin, vinculin, TNKS-1, PARP1.
2-In figure 6 the TNKS-1 inhibitor XAV939 does not completely inhibit the “PARP epithelial adhesion belt”. Given the high potency of XAV939, this result does not seem to agree with the author’s hypothesis. The authors should perform additional experiments in which XAV939 is added only after seeding of the cells to see if the PAR belt disappears later.
3-The authors showed that “Figure 3 highlights the fact that PAR is associated to sub membrane domains only in the proximity of a neighbor cell.” But PAR staining in figure 2 (F) and in figure 3 (B) look different. They used same antibody for the same specimen, the authors must explain why PAR staining is different?
Minor issues:
1-The authors should explain more clearly why they choose 3-AB, Olaparib, PJ34, XAV 939.
2-In material and methods, it is written “In all cases, cells were fixed 5 h after treatment initiation.” Is this means all treatment (PARP inhibitors and EGTA) were for 5 hours? In the “Results and Discussion” part it is specified “accordingly, nuclear PARPs inhibitor (Narwal et al. 2012) Olaparib (250nM, 6 days)”. Is the treatment with Olaparib for 6 days? It is confusing and the authors must explain the protocol more clearly in the material and method part.
3-In figure 2, treatment with 3AB and PJ34+3AB showed differences in actin structures, authors must discuss and explain this in the results and discussion part. According to Riffel et al, 2012, PJ34 exhibits at least 30X higher potency towards PARP1 than to TNKS-1. Why then does PJ34 treatment result suggest TNKS-1 involvement?
4-Many panels of Figure 2 are not indicated in the text. The authors should give detail to which panel exactly they are referring to.
5-In the introduction part, the reference style (Virag 2013) is not similar like others. The authors should write it in similar way.
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In the manuscript, Laura Lafon-Hughes et al. have investigated the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO) and demonstrated that the existence of PAR in a novel subcellular localization and consistented with the view that such PAR may be synthesized by TNKS-1 . This work could be of interest in Poly-ADP-ribose (PAR) research area. However, some important concerns should be addressed:
In the manuscript, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO) and demonstrated that the existence of PAR in a novel subcellular localization. So, the pictures for the results are very important. But in the “Results”, legend, the FIGURE LEGENDS of pcture 4 are not matching with the pictures 4, especial for C and D. Please check them and correct them.
When suitably revised, this paper will make a very interesting contribution to the growing literature on the Poly-ADP-ribose (PAR) research area.
The manuscript requires editing in some areas. For example:
Line 210: "went along" is not scientific language
Line 231: IC50 values for TNKS-1 not shown
Also, the authors need to expand on descriptions and explanations of experimental findings in the results section, as well as the conclusions. These are important edits that can easily be accomplished upon revision.
The authors present very impressive visual images of their findings. However, more clarity is required for some figures:
Fig. S3: was there really 6 days of olaparib treatment?
Fig. 2: what was the total length of time for PARP inhibitor treatments?
Fig. 4: confusing--is red staining for actin or vinculin? If actin, how was vinculin detected and visualized?
Fig. 5: This is impressive data, with striking images. Please expand explanations in lines 211-214. What do the arrows and arrowheads indicate? What can be concluded from this figure?
The primary concern is that XAV939 also inhibits tankyrase-2 and the authors repeatedly implicate tankyrase-1 in the findings. The authors need to clearly acknowledge that TNKS-2 may be a possibility in the results and conclusion sections.
Also, a minor point is that it is well-known that different PAR antibodies detect different types of PAR. Most were engineered to detect complex and long PAR that results from DNA damage. Since all the anti-PAR antibodies used were polyclonal, it may help the explanation and justification of the results by discussing how each antibody was raised. Via BSA-hapten? With long or short PAR?
This manuscript presents very impressive visual images that uncover novel roles of poly-ADP-ribose (PAR) in the mediation of cytoskeletal functions. However, the hypothesis that TNKS-1 is the enzyme responsible for this is a bit too premature. Also, the justification for the use of a particular fixation technique and a particular PAR antibody is a bit confusing as well. But the data is unique and does allow for new contributions to the fields of PAR and cytoskeletal dynamics. With minor revisions in text and editing, i.e. better explanations and justifications, acknowledgement that TNKS-1 may not be the primary enzyme responsible, this manuscript will be suitable for publication in PeerJ.
The manuscript meets the criteria of basic reporting. The chapters are well structured and can be followed logically as well. Relevant prior literature is appropriately referenced.
Minor revisions for the authors:
-Fig. 5, 6 is not mentioned in the text. For a proper conclusion they should be featured.
-The abbreviation of "PFA" is not introduced in the text.
-Supplier, catalog number and animal species is missing for H10 clone anti-PAR antibody.
-anti-chicken anti-antibody is not mentioned on the list of secondary antibodies.
-The type of antibodies (mono-, or polyclonal) should be stated because this could be linked to the different recognition properties of the used antibodies
-The paragraph starting from line 148 in the Results section is ambiguous. Avoiding the permeabilization step allows to study only extracellular antigens not intracellular. Therefore I suggest changing the word intracellular to extracellular in the sentence: ...we decided to check the intracellular nature of... For this reason the last sentence should also be deleted because it states an evidence in this context.
-In line 174 the authors mention Olaparib treatment (250nM, 6days) which also leads to misunderstandings. In the methods section only 5 hours treatment is mentioned. This should be clarified.
-Such comparison of IC50 values in line 174 and line 181 is not widespread in the literature. Nevertheless, it also leads to a wrong conclusion. For Oliparib the authors' comparison claims that it is a more potent inhibitor on TNKS-1 and for PJ34 that it is more potent on PARP-1. I suggest using the exact IC50 values for comparing the affect of the drugs on the enzymes.
-Using a sub-heading before the paragraph that starts from line 176 could make the whole text more transparent. The results discussed from here are coming from treated VERO cells but the chapter start from "Untreated Vero cells"...
-Figure 5 legend should be changed as follows: ...cytochalasin D, (I-L)...
-Figure 4: which color is for vinculin?
The research described in the manuscript is relevant and meaningful. The experimental design meets the technical standards and the methods are described with sufficient information.
Minor revisions for the authors:
-Using a positive control for long branched PARylation (e.g.: oxidative stress for the cells) would clarify the negative results for H10 antibody. The authors properly explain this phenomenon with the preference of H10 antibody for long PAR chains but an experimental observation would undoubtedly demonstrate the validity of that assumption.
The findings of the manuscript are very interesting and worth publishing. The conclusions about the existence of a "PAR belt" are appropriately stated and limited to those supported by the results. However these observations should also be supported by independent methods. For example a fractionation experiment would clarify the specificity of the antibodies. Proximity ligation could also confirm the existence of a peripheral, actin associated PARylation process. An immune-independent method would be the use of tritium labeled NAD for tracking compartment specific PARP activity.
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