API-2-Induced Cell Migration Is Overcome by Small Molecular Approaches Inhibiting β-Catenin

Round 1

Reviewer 1 Report

Material and Methods 2.2: There is not any immunoblotting result in the paper.

Fig.2 The expression of β-catenin and FOXO3a should be quantified. Moreover, the results couldn’t support the conclusion that the synergistic inactivation of AKT phosphorylation by API-2 and KY1022.

The lack of clarity on the number of independent repetitions performed

The scale bars should be added to all the HE and IF figures.

The manuscript needs to be carefully checked for typos and language editing.

Discussion was not written appropriately.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript, Dr Yonghyo Kim and colleagues analyze the effect of the β-catenin destabilizer, KY1022, on cell migration and viability of CRC cells harbouring APC and KRAS mutations. The manuscript in general is barely sufficient and the data are very preliminary, both considering how the experiments are conducted (basic techniques) and observing which assays they have carried out. Furthermore, the authors use only a cell line, the LoVo cells and it is not sufficient for deducing conclusions. Therefore, the authors would give less emphasis in the introduction.

 

Materials and methods:

-        Antibody codes should be added.

-        Phalloidin staining. Generally, cytoskeleton rearrangements are cellular phenotypic changes that occur in a few minutes (30-60 minutes). The authors should test these new conditions.

-        In the paragraph “wound healing” the authors write: “LoVo cells were coated with collagen (500 μg/mL) were coated in 6-well or chamber.” It is not clear. Please, explain better. I think that the authors are confusing wound healing assay with transwell assay. Additionally, are the authors inhibiting the proliferation? It is an important point.

-        The authors should revise the whole “mat and met” section.

-        In relation to paragraph 3.1 in the MAT and MET section, a specific section indicating what software has been used lacks.

 

 

Results:

- In figure 1, please write a reference to the software used for the analysis.

-        Line 154: please add a reference

-        The authors write: “While API-2 slightly decreases the level of nuclear β-catenin, KY1022 effectively decreases the nuclear β-catenin with or without API-2 treatment.” Why do they decrease the nuclear beta catenin levels? Is it in the cytoplasm? Is it degraded?

-        Please add negative controls for IF staining A graphical analysis should accomplish the IF figures.  In addition, a western blot for beta catenin analyzing the different cellular fractions (Nuclei, cytoplasm) should be added.

-        The authors must perform also transwell assays (coated with collagen and Matrigel) inhibiting the proliferation for analyzing migration and invasivity.

 

In conclusion, the authors should re-write the entire manuscript, add experiments performed in another cell line and follow the suggestions of the reviewer

 

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

All my concerns have been addressed in detail.

Reviewer 2 Report

The manuscript Is Just a Little Better. The authors have not employed big efforts.

Nevertheless, in this form Is possibile to follow the whole manuscript