The Complex of p-Tyr42 RhoA and p-p65/RelA in Response to LPS Regulates the Expression of Phosphoglycerate Kinase 1

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the article entitled “The complex of p-Tyr42 RhoA and p-p65/RelA in response to LPS regulates the expression of phosphoglycerate kinase 1, leading to tumorigenesis”, the authors aimed to uncover the molecular mechanism through which p-Tyrosine 42 RhoA, in conjunction with NF-κB, would promote tumorigenesis. They induces inflammation by using LPS and observed that p-Tyrosine 42 RhoA co-immunoprecipitated with p-Ser 536 p65/RelA subunit in NF-κB in response to LPS. Moreover, both p-Tyrosine 42 RhoA and p-p65/RelA translocated to nucleus, where they formed a protein complex associated with the promoter of phosphoglycerate kinase 1 (PGK1) and regulated the expression of PGK1. In addition, p-p65/RelA and p-Tyr42 RhoA co-immunoprecipitated with p300 histone acetyltransferase. They also verified that PGK1 exhibited interaction with β-catenin, PKM1 and PKM2. By using a specific si-PGK1 they saw a reduction in β-catenin and phosphorylated pyruvate dehydrogenase A1 (p-PDHA1) levels, and found that PGK1 phosphorylated β-catenin at Thr551 and Ser552 residues. They concluded that PGK1 may play a role in transcriptional regulation alongside other transcription factors.

 

This data provide new insight into the possible treatments in order to counteract tumorigenesis.

 

Data reported are interesting and could be taken in consideration to provide new potential basis for the treatment of tumors. However, the paper can’t be considered for publicaton in its present form but it needs to be revised. I reported here my comments:

 

The authors should add an ABBREVIATIONS Section to the manuscript in order to clarify the meaning of such acronyms reported within the text.

 

There are a lot of text errors which have to be fixed within the manuscript. Some examples are “lipopolysacchrtide” instead of “lipopolysaccharide” (lane 46), “resulrting” instead of “resulting” (lane 50), “choangiocarcinoma” instead of “cholangiocarcinoma” (lane 497), and so on.

There is also a truncated sentence at lane 59 (“In response to TGF-β1, 58 RhoA/ROCK, in turn, phosphorylates IKKβ in response to [8]”), which has to be reorganized and completed. Please check the whole manuscript to fix them.

 

In the MATERIALS and METHODS Section, sub-paragraph named “2.2. Cell cultures”, the authors reported that they used three cell lines, the human embryonic kidney cell lines HEK293T, the mouse breast cancer cell lines 4T1 and the murine macrophage cell lines RAW264 to validate their results. Why did they chose these three heterogenous cell lines? Please clarify that.

 

In the METHODS Section, sub-paragraph named “2.7. Small interference siRNA transfection”, the authors reported to have used a specific small interfering RNA against RhoA (sc-91064), p65 (sc-29411), PGK1 (36216), ROCK2 162 (S36433), with respect to an unspecific control si-RNA (sc-37007). How many specific siRNAs directed against the reported targets they were used (by considering also setting experiments)? I think it would be better to repeat the experiment with at least another siRNA, that specifically targets the reported targets, in order to verify the reproducibility of their data and to avoid off targets effects, or at least the authors have to indicate if they were tested more than one siRNA and then the best resulted interfering molecules were later used. Please report the siRNAs sequences used. Please clarify.

In the MATERIALS and METHODS Section, sub-paragraph named “2.8. Site directed mutagenesis”, the authors reported that “Additionally, GST-beta-catenin WT, S502A, 169 T551A and S552A mutants were generated through the use of a site-directed mutagenesis kit (15071)”. What does it means the number in round brackets? Please clarify that.

 

 

In FIGURES 1F-G-H-I-J-K-L, 2A-B, 3A, 4B-C-D-E-F-G, 5F and 6E-F-G-L, please add the histograms representing revealed protein/control protein ratio.the molecular weight (kDa) for all of the revealed proteins to the WB panels.  Please also provide raw images of the whole membrane from the biological replicates.

 

In general, figures are too little and need to be enlarged. The numbers indicating protein revealed/housekeeping protein ratio make the images confusing and have to be substituted by more representative histograms.

 

FIGURE 4H is not reported but it is commented within the text (In the subparagraph “3.4. LPS induces PGK1 through p-Tyr42 RhoA and p-65” of the RESULTS Section)

 

Cell proliferation assay is reported to be used in some experiments but its protocol is not reported in the MATERIALS and METHODS Section. Please provide it.

 

 

In conclusion, in this paper, the authors reported some interesting data about possible future treatments in order to counteract tumorigenesis.

The data are potentially interesting but need to be improved in order to be taken in consideration for publication.

The paper potentially reports interesting scientific data but I think that it needs to be revised and improved in its content (and also in its english style) in order to meet the journal aims and be considered for publication.

 

Comments on the Quality of English Language

Many text errors to be fixed

Author Response

Thank you for your critical and careful comments. We responded to each comment in attached file. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The introduction should be rewritten in such a way as to include a better description of NFkB pathway activation of the Rho.

Fig 1H. The text states that when knocking down Rock1 or Rock2 p5: l 229-230. P-p47 goes down. However, if one looks carefully at the figure, it is evident that Rock1 knockdown is not successful which can be seen on the blot and the densitometric measurements made and calculated by the authors. Authors should make efforts to obtain a successful knockdown of this protein to sufficient levels of at least 80% of the control.

Problematic is also the knockdown of Rho an on Fig. 4d according to the blots and densitometry. In both these cases authors should use more efficient siRNAs in order to convince the reviewers of their claims.

Fig 6B, it's very strange results that HEK 293 cells which are motile did not close the wound even after 48h. This contradicts many publications one of which is referred-PTPN6 regulates the cell-surface expression of TRPM4 channels in HEK293 cells October 2018 European Journal of Physiology 470(10) DOI:10.1007/s00424-018-2161-9. The authors should explain why their results contradict other researchers' observations.

 

Minor comments:

P1: L 34 plays are repeated.

P2: L 59 incomplete sentence.

P2: L61 unclear sentence

P2: L 63 translocate TO the nucleus

P3: L 118 h should be hours

P3: L131 added to instead added by

P4: L147 This sentence is unclear

For all densitometry graphs vertical line should be (Fold change relative to control)

Paragraph 2.7: The description of siRNA transfection protocol is unclear. Was transfection done for 48h or 4-5h? At what time point after the start of transfection were all assays performed? This procedure should be written very clearly.

Paragraph 2.10. The description of the staining procedure is also unclear. Authors should indicate exactly which were primary antibodies????

Paragraph 2.11: -Different font

In all confocal images, a scale bar should be included.

Maybe it will be a good idea for all confocal images to be presented in bigger sizes and only merged images with a proper color indication.

 

Comments on the Quality of English Language

Authors should thoroughly check their manuscript for spelling mistakes and missing words and phrases. 

Author Response

Thank you so much for your careful and critical comments. We responded to each comment in attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, authors explored role of RhoA and p65/RelA in regulation of PGK1 in response to LPS stimuli, and their mechanistic acts. Their findings unveil the interaction between RhoA, p65/RelA, and p300 and the transcriptional function of this complex for PGK1 expression. Furthermore, this study also observed that PGK1 phosphorylated b-catenin and cell migration. Overall, this study has interest and merit. The study is well designed and their experiments have been properly conducted. The conclusions can be fully supported by their results, and the manuscript is satisfactorily prepared. Before it could be accepted for publication, some minor issues remain to be addressed.

1. In title, authors express that LPS-triggered RhoA/RelA/PGK1 axis may lead to tumorigenesis; however, their results lack direct evidence to support or reflect this purpose. It is suggested to modify the title to more reflect the major aim of this study.

2. Y27632 may also inhibit PKC, the dose/concentration of Y27632 used for inhibition of ROCK (fig.1C, 1I) should be indicated.

3. There are obvious typos and improper formats. In addition to incorrect formula of chemicals, it includes typos such as #50 “resul;ting”, #106”CO2”, #111, #126” Na3VO4”, #161”CO2”, #469” O-glcaacylation”, #406”through”(repeated); redundant abbreviation such as #115”PVDF” (been defined in #84), #133”SDS-PAGE” (been defined in #114), #174”RT” (been defined in #137); and inconsistent word style in #201-203. Authors should correct these errors.

4. #216-217, P<0.05 is considered as statistically significant, surely P<0.01 and P<0.001 are significant. It is recommended to modify this sentence.

5. Fig. 5A, clearer image is needed to evaluate the expression of PGK1 in response to LPS.

6. Fig. 5E, also in #454-455, the findings showed that pY42-RhoA interacted with p300; however, there is no evidence to show the histone acetylation is increased in response to LPS.

Comments on the Quality of English Language

The English is fine, except for some typos and inconsistency.

Author Response

Thank you so much for your critical and constructive comments. We attached the file of response letter. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In the 2nd version of the article entitled “The complex of p-Tyr42 RhoA and p-p65/RelA in response to LPS regulates the expression of phosphoglycerate kinase 1, leading to tumorigenesis”, the authors addressed all my comments.

 

I think the manuscript may be considered for publication in its present form.

Reviewer 2 Report

Comments and Suggestions for Authors

All my concerns were addressed.