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Thanks for the revision of the manuscript, which can be now accepted.
[# PeerJ Staff Note - this decision was reviewed and approved by Pedro Silva, a PeerJ Section Editor covering this Section #]
The manuscript which you submitted to PeerJ, has been re-reviewed. The reviewers have recommended publication pending minor revisions. Therefore, I invite you to respond to the reviewers' comments at the bottom of this letter and revise your manuscript accordingly.
Although some defects cannot be made up due to the outbreak of new coronavirus pneumonia in China, this manuscript is generally acceptable.
The authors have included the clarification and additional information in the Introduction and Materials&Methods section of the manuscript as requested. Figure legends were also updated as requested. Please note that the legend of Figure 3 was updated in the text part of the manuscript, but not yet in the separate Figure 3 file.
The Materials&Methods section has been updated and includes now all relevant information with sufficient detail. Missing information (e.g. western blotting procedure and references to used antibodies) is added.
Importantly, the confusion concerning the number of replicates per experiment and the number of independent experiments is solved. Missing raw data are made available and are better annotated.
However, it seems that the raw data for the invasion assay may not be entirely correct. When looking at the mean number of invaded cells with 100ng/mL CXCL12 under normal conditions, this number seems to higher than represented on Figure 2A of the manuscript. Please clarify.
The authors commented on Question 3 (the reason why siELMO2 by itself already gave a decrease in the number of migrated cells) and Question 5 (the interplay between the expression levels of ELMO2 and GNAI2) in their rebuttal letter, though they did not include these comments in the manuscript itself. Although I don’t consider this as a major issue, it could have enriched the discussion section.
As the authors suggest themselves, few small additional experiments (e.g. testing the specificity of siRNA towards ELMO2 by also including ELMO1 and ELMO3 samples) may have further strengthened the data, but I understand that the outbreak of Corona virus may have hampered this.
The manuscript has certainly improved in clarity and readability and now contains sufficient experimental detail.
The reviewers have recommended publication pending major revisions. Therefore, I invite you to respond to the reviewers' comments at the bottom of this letter and revise your manuscript accordingly.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in an appropriate rebuttal letter, and please ensure that any edits or clarifications mentioned in the rebuttal 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 it is safe to assume that your readers will have the same question) therefore, you should ensure that the manuscript itself also reflects the responses you make in the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
The paper is well written overall. The references used are sufficient.
The figures are prepared professionally and the raw data is present as well, however it is not always clear how this relates to the experiments. For example the F actin content looks like the raw data is given, however the figure shows relative F actin content without any indication how this was calculated. Without labelling which data point belongs to which condition, it is impossible to see whether the derived curves are reflecting the raw data. More information about the raw data and the actual manipulation and calculation of the data is needed
For the adhesion data for the PANC cells, there is an error in the calculation for si at 0ng in the raw data, it is not a huge difference, however that does not fill me with confidence that all calculations are correct
Overall the experimental design is good, however in particular the chemotaxis assay is lacking details in description of methodology. There are no details about what type of plates are used, whether there are transwell or others. Also, there is no description of how the adherent cells were counted, adherent cells do not migrate through a membrane, they will stick to the membrane, but the methodology described gives no details about the procedure.
The data looks robust, the migration data is very tight, which is a bit surprising since all experiments were done as an n=3 and woundhealing assays in particular show some variety of response, but the raw data provided supports this.
I don't like seeing the input bands in Figure 3 used twice, if all experiments are done on the same membrane, it is valid, however I'd appreciate if the input is only shown once.
It would be helpful to have the raw data annotated better, so it can be seen when each experiment was done and also how relative values were calculated.
In lines 254-256, please list all the reference articles about ELMO3 study.
In order to give the reader a direct impression, the author is suggested to present a picture of immunofluorescence about F-actin Polymerization. Please refer the paper of PMID: 30345300
(1) The authors should explain why PANC-1 and AsPC-1 were chosen in this study. In Fig 1(A), it seems ELMO2 revealed a weak protein expression in PANC-1 cells. In addition, both two cell lines presented low invasiveness in normal condition in Fig 2(A). I was wondering if other suitable cell lines can be used such as Capan-1 cell. Or the author should optimize the experimental conditions.
(2) I suggest that the authors make a statistical analysis of whether silencing ELMO2 affects cell chemotaxis, migration, invasion and F-actin polymerization without CXCL treatment, at least make a mark on the Fig. 1 and Fig. 2.
Invasion and migration are important reasons contributing to the death of pancreatic cancer patients. In this manuscript, the authors thought ELMO2 could promote pancreatic cancer cell chemotaxis, migration, invasion and F-actin polymerization. Furthermore, they also found chemokine CXCL12 triggered Gαi2-dependent membrane translocation of ELMO2. Generally, this study is critical in exploring the role of ELMO2 in pancreatic cancer. However, these issues should be concerned by the authors.
Line19: “knockdown” instead of “knockout”
The role of the ELMO protein (ELMO1, ELMO2 and ELMO3) family is described (line 40 - 55). It is however, not clear why the authors specifically focus on the role of ELMO2 in pancreatic cancer (as stated in line 55), and not on the role of ELMO1 or ELMO3.
For instance, is there any evidence/suggestions from the literature that links ELMO2 with pancreatic cancer? What about redundancy between the function roles of the three ELMO isoforms? I suggest the authors elaborate a bit more on this topic and clearly state why they choose to study ELMO2 in the specific context of pancreatic cancer.
The authors state that ELMO family members have been implicated in a variety of malignant cancers including glioma, breast cancer etc (line 52-53). I think it would be of interest to mention for the different types of cancer if these data were obtained from in vitro studies or in vivo studies.
Description of the Materials and Methods:
1. Line 105: western blotting is mentioned. However, the protocol for western blotting (including a reference to the antibodies used), is not included in the manuscript. Please include.
2. Line 106 - 109: ELMO2 siRNA sequences are shown. It is not clear which of the three siRNAs is actually used throughout the experiments. Did the authors use only one siRNA to knockdown ELMO or did they use a combination of all three siRNA at the same time? Also, how specific are these siRNAs for ELMO2 (Is there any change of also targeting ELMO1 and ELMO3 with these siRNAs?)
Line 118: Please describe how cell the lysates were prepared.
Line 124: Please better specify the used antibodies (e.g. add catalog number)
What primary and secondary antibodies were used in the study.
Line 148: The number of migrated cells in the chemotaxis assay seems rather low (Figure 1). Please mention how many cells were added to the upper compartment? Also, include a reference for the type of transwell plate(s) that is used in this experiment.
Cell Invasion assay:
Also here, it is not mentioned how many cells were used and when they were added to the plates. Also include a reference for the type of plates that were used.
Line168: How many cells in 100µL?
Line 184: Add the number of replicates per experiment.
In Figure 1 and Figure 2 it is stated that all data are represented as the mean of three independent experiments. How many replicates were performed per experiment? Also, the raw data of these experiments are made available as supplemental files. However, it seems that the data of only one (out of three) experiments is given for each type of experiment. Can you clarify this, and if needed, add the missing data?
In Figure1B and 1C: Does the “siELMO2” condition refer to cells in which siELMO2_1, siELMO2_2 or siELMO2_3 siRNAi was used? Or a combination of all the siRNA sequences?
It appears in Figure 1B that the siELMO2 condition by itself (thus, without CXCL12 stimulation, 0 ng) already gives a decrease in the number of migrated cells. Is this decrease statistically significant? If yes, can you speculate about a possible explanation for this?
Line 208-209: “CXCL12 generated a transient F-actin accumulation in PANC-1 and AsPC-1 cells, in line with previous findings.” Please, add a reference to the literature for this statement.
In Figure3A and 3B: Please specify what “Input” and “IP” means in the figure legend. My interpretation is that when PANC-1 cells are transfected with GV362-ELMO2, a clear (and expected) increase in expression of ELMO2 (70kDa) is observed in the input sample compared to non-transfected (normal) cells. It seems, however, that also the expression of GNAI2 has increased in the GV362-ELMO2 condition compared to normal, which is rather unexpected. Is this interpretation correct? How can this be explained?
Similarly, when overexpressing GNAI2 it seems that also the expression level of ELMO2 increases?
Figure 4A: Adding the DAPI stain to all panels could help in giving a better visualization of the data.
In Figure 4A and Figure 4D: Where do the arrows point at? Pleas describe in the figure legend.
This manuscript is well-written and data are clearly presented and structured. It is, however, not immediately clear what the novelty and importance of the work is, given that the role of ELMO proteins in GPCR-mediated chemotaxis has been described previously. The novelty and importance of your work should be more clearly stated in the introduction part.
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