Peer-reviewed by 2 reviewers with median rating of 15.5/20. Review process was triple-blinded.
Round 1 (16.5/20)
Round 2 (15.5/20)
Conceptual advance and Impact7
This is an interesting paper describing the effect of PEI transfection on the integrity of the endosomal system. As predicted, PEI induces loss of endosomes without affecting lysosomes number. The authors then developed a new protocol to decrease the damage to the endosomal system without compromising on the transfection efficiency. This is what will be of interest for the community. The paper is also technically very good. The only issue is that I cannot see the methods section. This should be addressed before publication.
I cannot see the methods section.
Conceptual advance and Impact6.5
This is an interesting study despite some technical limitations. It will be of interest to researchers who study the endolysosomal system using transient transfections, and has some important implications for all researchers who use PEI, or PEI-based transfection reagents, to overexpress proteins in cells. The problems that require addressing are: 1. Data presentation issues (graphs), 2. The use of excessive amounts of Effectene vs normal amounts of PEI.
A. The graph should have SD not SEM, ideally it should be presented as a "beeswarm graph" to visualize the variability - is the difference attributable to a small proportion of very large endosomes in PEI-treated cells, or are all endosomes slightly enlarged. One cannot distinguish between these possibilities by this graph. Also, do all endosomes contain PEI? If not, are the ones that do contain PEI significantly larger than the ones that do not? Is there evidence of endosomal rupture on any images? Are there "free-floating" PEI particles outside of endosomes, which would be predicted from the "rupture" hypothesis? All these are relatively simple questions that could be addressed using already collected data.
B. Use "untransfected" or "control" instead of WT - WT implies that there were mutants involved. What happened to the error bars in the control (black bars)?
C. Should the total Galectin fluorescence change in response to lysosomal rupture? I would expect the total fluorescence to stay the same and instead the number/size of puncta to change? How is it possible that relocation of the protein changes the total fluorescence/surface area? The results section talks about the size and number of punctae, but this is not shown in the graph.
Results & Discussion
1. typo: ration
2. typo: occures
3. What would happen if you used 5x the recommended dose of PEI - would that cause lysosomal leakage, similarly, if you use Effectene at low doses (1x), does that cause visible lysosomal rupture? You cannot compare overload with one reagent vs. low doses of another. It's perfectly conceivable that PEI causes leakage of a limited number of lysosomes and that you cannot obtain a significant difference just because the effect is weak at these doses.
4. typo: fluorescentpunctae