Dear Editors, Attached is our revised manuscript by Qi-Chao Xie*, Yu-Peng Yang (5171394981081306). Thank you and the reviewers for the positive comments on our manuscript. We are pleased to revise our manuscript based on these suggestions, and all the changes of our revised paper are in red. Responses to the reviewers: 1. Authors showed the anti-cancer properties of physcion 8-O-β-glucopyranoside (PG) isolated from Rumex japonicas Houtt. on A549 cell lines . This was achieved through modulation of cell cycle and induction of apoptosis in A549 cell line. However, the title of the manuscript is inappropriate it mainly focus on anti-proliferation of PG in A549 cells via cell cycle arrest and apoptosis. Response: Thank you for your good suggesting. We have changed the title to “Anti-proliferation of physcion 8-O-β-glucopyranoside isolated from Rumex japonicus Houtt. on A549 cell lines via inducing apoptosis and cell cycle arrest”. 2. The language throughout the manuscripts still needs improvement. Response: We have revised our manuscript seriously based on your comments. 3. Change the “anti-tumor” to “anti-proliferate”. Response: Thank you for your good suggesting. We have changed the “anti-tumor” to “anti-proliferate” in our revised manuscript. 4. In introduction section, please place Figure 1 under results section. Response: Thank you for your good suggesting. We have revised our manuscript and placed Figure 1 under results section. 5. In methods section (Page 6), the authors did not mention what concentration Response: We have added the concentration of PG in methods section (Page 6) of our revised manuscript. The cells were treated with 20, 40 and 80 μg/mL of PG for 24 h, or treated with 80 μg/mL of PG for 12, 24, 48 h, respectively. 6. In methods section (Page 7), the authors did not explain how fold changes were determined in western blot analysis. Response: Fold changes of protein levels were determined using Bio-Rad quantity one software and compared to the protein expression of β-actin. We have revised the expression of fold changes in methods section (Page 7) of our revised manuscript. 7. In results section (Page 7), the authors determined IC50 values of total extracts of R. japonicas on six different cancer cell lines at 24 hr. Is there any specific reason why 24 hr of post treatment was chosen? Response: We determined IC50 values of total extracts of R. japonicas on six different cancer cell lines at 48 h, not at 24 h. At this time, the cells are in the logarithmic phase. 8. The subheading in identification of compound from tested plant should be R. japonicas and not P. scandens. Please correct this. In addition, the authors should present the identified compounds from R. japonicas in table forms and present the structure of PG (Figure 1) under this section. Response: Thank you for your good suggesting. We have changed “P. scandens” to “R. japonicas”, placed Figure 1 under results section, and added the identified compounds from R. japonicas in table 2. 9. In results section (Page 8), the authors mentioned concentration of PG at 20, 40 and 80 µg/mL were selected based on effect of PG on A549 cell viability. Please explain this. Response: We used 5, 10, 20, 40 and 80 μg/mL of PG to investigate the anti-proliferation effect of PG by MTT test. The IC50 was 53.01 μg/mL at 24 h and 27.31 μg/mL at 48 h, respectively. So we have chosen three concentration gradients (20, 40 and 80 μg/mL) which are less than, closed to and higher than IC50 value for subsequent experiment. 10. In results section (Page 9), the authors showed that PG induced G2/M phase arrest when cells were treated at 80µg/mL at 48 hr. However, author should use a positive control to show the validity of the findings. In addition, the authors should perform a time-dependent study to observe the gradual accumulation or distribution of treated cells. Besides, it is also noticeable that there is a significant increase of PG40- and PG80-treated cells arrested at S-phase compared to untreated cells. What could be the reason for this observation? Response: Thank you for your good suggesting. We have performed a time-dependent study (0, 12, 24, 48 h) to observe the gradual accumulation or distribution of treated cells in figure 3C on our revised manuscript. PG induced cell cycle arrest at G2/M phase in dose-dependent manners. However, the PG did not show dose-dependent effects on S phase arrest. The reason may be that different doses of PG show different effects on cell cycle distribution. 11. In results section (Page 10), authors should use a positive control to show the validity of the findings where PG induced apoptosis in A549 cells. In addition, the authors have mentioned by adding Ac-DEVD-CHO could reverse the apoptosis in PG-treated cells. But from the results (Figure 5B), addition of Ac-DEVD-CHO in PG-treated cells do not completely reverse the apoptotic effect of PG and this indicate that PG could induce apoptosis using caspases-dependent pathway. Response: Thank you for your good suggesting. Our present investigation of PG against anti-proliferation of cancer cells supplied a basic and valid evidence for future study of PG, and we think our results were enough to demonstrate the anti-proliferation activity of PG. Furthermore, the further investigations of the PG are continuing, and we would rather to show the further systemic results with positive drugs to you in the future. The inhibition rate of 20, 40, 80μg/ml of PG is 35.19%, 60.33% and 63.39%, respectively. When A549 cells was pretreatment with Ac-DEVD-CHO (a caspase inhibitor), the inhibition rate of PG significantly decrease, it is 16.72%, 28.02% and 29.26%, respectively. The results demonstrated PG in combination with Ac-DEVD-CHO could partly reverse the anti-proliferate effect compared to PG-alone treatment group. 12. In discussion section, the content is too brief and superficial. Response: We have revised the discussion section of our manuscript. 13. In Figure 5A, axes of the scatter plots should be labeled accordingly. Level of interest: An article whose findings are important to those with closely related research interests Response: Thank you for your good suggesting. We have added the axes of the scatter plots in Figure 5A. Best regards, Qi-Chao Xie (PhD.) Department of Oncology, the Second Affiliated Hospital, Third Military Medical University, 183 Xinqiao main street, Chongqing, 400037, China Phone and Fax: +86-023-68774050 E-mail: [email protected] (Qi-Chao Xie).