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All critiques are adequately addressed and revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Pedro Silva, a PeerJ Section Editor covering this Section #]
Satisficed with the author's answer.
experimental design well
Nampt plays important roles in oxidative stress-produced decreases in NAD+ levels and cell survival. The findings indicated the roles of Nampt in maintaining NAD+ levels and cell survival under both basal and oxidative stress conditions.
The authors respond to all my queries satisfactorily
The authors respond to all my queries satisfactorily
The authors respond to all my queries satisfactorily
The authors respond to all my queries satisfactorily
Please address the concerns of both reviewers and amend your manuscript accordingly.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
no comment
no comment
no comment
In this study, Zhou et al showed that Nampt plays significant roles in both the NAD+ synthesis and survival of the cells under basal conditions, their study indicated that oxidative stress can decrease both the mRNA and protein levels of Nampt, which indicated a novel mechanism underlying the NAD+ deficiency in aging and multiple neurological disorders. Overall, these findings suggesting a new mechanism underlying oxidative cell death.
There are a few concerns that the authors should address, which are detailed below.
1. In this study, the author used differentiated PC12 cell as a cell model, however, the cell culture condition seems not a differentiation condition, please clarify. Does your differentiated PC12 cell have neurite outgrowth?
2. Fig 1 B and C legend, “PC12 cells were treated with 2, 5, 10, 20, and 50 nM FK866 for 24 h rs.” please remove 2, 5, since your figure did not include these two concentrations.
3. Does author have Supplementary figure legend?
4. Treatment of the cells with 0.1 or 0.3 mM H2O2 led to significant increases in the Nampt mRNA level of the cells at 12 hrs, in contrast, H2O2 led to significant decreases in the Nampt mRNA level of the cells at 20 hrs after the H2O2 treatment. Does author have any explanation?
This is an interesting work that intends to elucidate how oxidative stress regulates the expression and protein levels of the enzyme NAMPT, involved in the salvage pathway of NAD synthesis.
The abstract needs improvements, the objective of the work is not clearly expressed, also the results.
The introduction has the elements necessary to understand the work, however the summarizing of main results do not follow the sequence of the results.
The methods are adequate to respond the objective of the work. However, some interpretation of the data seems to be mistaken.
For example, in figure 1A the authors show that FK866 induces a decrease in the NAD levels, but the effects are similar between all the concentrations tested. However, there is an increase in cell death concentration-depended, How the authors explain these results? It's possible that the increase in cell death is independed of reduction in NAD levels?
The make the data more robust, may include the data of cell death in response to H2O2 with the NAMPT siRNA
In figure 2, the authors show a reduction in the expression and protein levesls of NAMPT induced by oxidative stress, but what is the impact of this reduction in NAD levels?
In figure 3, the authors included a inductor of NAMPT activity, but what is the effect in NAD levels?, also the authors should include the flow citometry assay for this experiment.
Figure 4, its show the effct over ATP levels, however the reduction could be related to the reduction in cell viability induced by H202. It´s important to define what do you want to respond with this experiment.
According to the description in the text of the figures, the experiments are pooled from three independents experiments, with an n=6-8. However, to be exact the n =3 with replicates, thus to perform the analysis you must calculate the average of your replicates and perform the statistical analysis with an n=3.
The manuscript presents interesting data regarding the regulation of NAMPT by oxidative stress. This result is the most interesting of the work, however, the authors do not profundity in the mechanisms associate with this regulation. It´s important to unveil if the oxidative stress induces degradation of NAMPT or a reduction in the traduction of the protein. Also, the use of a precursor of NAD+ such as nicotinamide riboside would improve the quality of the work.
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