SLC7A11 and G6PD in Decidualization: A Critical Review and Suggestions for Improvement

Reviewer #1: In this study, authors aim to demonstrate the role of SLC7A11, which facilitates the cellular uptake of cystine, and G6PD-mediated PPP, which provides NADPH, in generating reduced glutathione (GSH) and the anti-oxidative stress potential during decidualization of stromal cells. While the topic is interesting, the study is too broad and not focused, and the evidences provided were far from enough for proving the causal relations to support the conclusion. The hypothesis here indeed includes two independent pathways that do not affect one another, although both are crucial for producing GSH, that is, SLC7A11-mediated cystine uptake, and G6PD/PPP-mediated NADPH production. In this term, it might be better to divide this manuscript into two. Specifically, my concerns are as follow.

1. Abstract should be rewritten. Abstract should be started with two to three sentences introducing the background of the study and then followed by the problem to be solved, results, and conclusion. In this manuscript, it is unclear which are known backgrounds and which are the novel findings. Furthermore, the authors stated that the PPP is crucial for stromal cells decidualization and the mechanisms underlying its role (which, indeed, could be deleted from the abstract as abstract should be compact), while the conclusion is that they elucidated the crucial role of SLC7A11-mediated GSH synthesis in stromal cells, without clearly stating the relation. This is confusing, as SLC7A11 is a cystine-glutamate transporter that mediates the exchange of intracellular glutamate and extracellular cystine, and the authors did not describe clearly the relation between SLC7A11-mediated cystine uptake, NADPH produced by PPP, and GSH synthesis. Moreover, while the authors stated the aim of this study, the problem to be solved was not stated clearly.

2. A thorough English editing is necessary, as some of the logics are confusing and the manuscript could be more compact. Furthermore, there are also some mistakes, such as the lack of a space between the period and 'PPP' (lane 103), and some wordings also need improvement. Also, the authors should pay attention to abbreviation. For abbreviations, full-name should be mentioned at their first appearance, and then abbreviations should be used afterwards. The authors had mentioned 'GSH' as the abbreviated form of 'reduced gluthahione' (lane 70); however, they again used the term 'reduced glutathione' (lane 116). This should be avoided. Other abbreviations, such as RT-qPCR (lane 195, should be quantitative real-time PCR (qRT-PCR), indeed), GPX, GSSG, etc. their full-names also should be mentioned at their appearance (lane 195 and lane 240, respectively). Please check the whole manuscript for similar problems.

3. There is no Ethical Statement. The authors had mentioned some related points in the 'Study Approval', however, these are not standard format and the information provided is far from enough. Ethical Statements for animal study and clinical samples should be separated. For animal studies, the source of animal, the facility for conducting the animal experiment, whether the animal study has been approved by an Ethical Committee for animal experiment (usually the Ethical Committee of the animal experiment facility), and whether the animal study has followed the protocol approved by the Ethical Committee for animal experiment should be stated. For clinical samples, the source of samples, the criteria of the samples, patients' informed written concerns, whether the study was approved by the Ethical Committee of the institution where the samples were collected, and whether the study meets the regulation of Declaration of Helsinki should be stated.

4. Lanes 150-153 and elsewhere, short interfering RNA should be abbreviated into 'siRNA' but not 'SiRNA'.

5. What it the antibody used for IHC? The authors did not state what staining they have done for IHC.

6. Figure 1A, the authors mentioned that these are the relative SLC7A11 mRNA levels. What are the controls used? If they compared the levels of SLC7A11 in DE groups to the NC groups (here, the full-name of DE and NC should also be stated in each corresponding Figure Legend), then why the level in NC groups where not 1? Furthermore, is there any time-dependency between the level of SLC7A11 and the length of treatment?

7. The orders of Figures 1B-F are confusing. As, according to the Central Dogma, mRNA is transcribed into protein, the results of qRT-PCR should come before western blotting. This also applied for Figure 2B, C, Figure 3A-D, etc. Furthermore, the authors should pay attention for the position of each sub-figures. It is confusing to have Figure 1C and 2C at the right site of Figure 1D, 2D, and 2E.

8. The logic of Figure 2 is very hard to understand and the evidences for stating that ROS decreased during decidualization while the GSH antioxidant system enhanced, and that oxidative stress impaired decidualization are insufficient. The authors should first show the evidences supporting decreasing ROS during decidualization, i.e., DCFH-DA, IGFBP1, glutathione reductase, GPX, and Bodipy staining. Then the authors should show that oxidative stress affected decidualization marker mRNA and western blotting. Furthermore, does IGFBP1 mRNA expression level showed hydrogen peroxide-dose dependency? Also, does ROS scavenger improve decidualization under hydrogen peroxide?

9. The level of PRL should be shown along with that of IGFBP1 at both mRNA and protein levels throughout the manuscript.

10. To proof that SLC7A11 is crucial for decidualization, rescue experiments using SLC7A11 overexpression vector and hydrogen peroxide/cAMP+MPA-treated cells should be performed. Similarly, experiments using ferroptosis inhibitors should also been performed.

11. Characterization of ferroptosis is still far from enough. Mitochondrial morphology, cell death rate (not by Annexin V/PI, as ferroptosis is different from apoptosis), etc. are needed (inhibitory experiment, induction experiment, and rescue experiment). Furthermore, the effect of SLC7A11 knockdown on cystine uptake should be shown.

12. Figure 5, again the orders and positions of the panels are confusing. Besides the order of western blotting and mRNA levels, NADPH should be placed before NADPH/NAD+ ratio.

13. The statement that 'These findings indicate that NADPH may exert a promotive effect on the process of decidualization' was over-concluded. From the increase of NADPH in cAMP+MPA-treated stromal cells, there was no any indication that NADPH could promote decidualization at all. Furthermore, from Figure 5E, it is not clear whether G6PD inhibition could reduce the level of decidualization, as -Actin levels also decreased significantly in cells treated with 5 and 10 M 6-AN.

14. Figure 5 only showed the correlation, but not the causal relation, between NADPH and decidualization. Furthermore, while Figure 5 showed that G6PD/PPP could affect decidualization (although the evidences was not sufficient, as the authors should at least overexpress G6PD and investigated its effect on decidualization), however, whether NADPH was involved remains unclear. NADPH is not the only product of PPP, and PPP could generate other products.

15. Figure 6 only showed that SLC7A11 affected ROS, GSH, and decidualization levels, and the effect of G6PD knockdown on ROS, GSH, and apoptotic levels. These are far from enough to proof that SLC7A11 and G6PD works synergistically in producing GSH and reducing stromal cell oxidative stress, and eventually, decidualization. Both pathways might affect decidualization, however, their causal relation and synergism needs further evidences. Is PPP the only source of NADPH for generating GSH in stromal cells? Experiments (all ferroptosis characteristics, all decidualization markers) using siG6PD/SLC7A11 overexpression and G6PD overexpression/siSLC7A11 should be performed.

16. I can't understand why the authors observed apoptotic rate here. The effect of SLC7A11, GSH, and erastin showed in previous figures indicated the possibility of ferroptosis. Thus, instead of Annexin V/PI staining, the authors should examine more ferroptotis characteristics, such as lipid ROS, mitochondria morphology, mitochondrial membrane potential, and cell death rate (PI single staining).

17. Figure 7, what is the relation between glucose depletion and decidualization? How about the role of SLC7A11 here? Also, what did the author means by Glc? Such abbreviations should be explained in Figure Legend, and abbreviations that make confuse and misunderstood should be avoided, it is hard to understand that Glc represents glucose depletion).

18. Lane 448, the statement 'NADPH and GSH (both were products of G6PD in PPP' is strange. First of all, G6PD does not produce NADPH directly. Secondly, it is improper to state that GSH is a product of G6PD and/or PPP, although NADPH is necessary for producing GSH.