Review Reports - Natural Extracts of <i>Alnus japonica</i> Induce BAK-Dependent Autophagy to Inhibit Liver Cancer Stem Cell Tumorigenesis

Round 1

Reviewer 1 Report

In this manuscript, Wuputra K et al. demonstrated that extracts derived from Alnus japonica (both fermented and non-fermented) reduced the proliferation of liver cancer stem cells through BAK-dependent autophagy. Although the data are rich and the results are interesting, several issues need to be addressed before publication.

Major points

１ What is the difference between the non-fermented and fermented extracts? The authors have listed the molecules contained in the non-fermented extract in Supplementary Table 1; however, no information is provided regarding the composition of the fermented extract. It would be helpful to know how much the compositional profiles differ between the two extracts. If any experimental data on the components are available, please include them. If such data are not available, this point should be clearly discussed in the Discussion section.In particular, in the xenograft experiment shown in Fig. 5, the fermented extract appears to reduce tumor size more effectively than the bark extract. Is this difference statistically significant? If so, the authors should discuss the possibility that the fermented extract contains additional or different active components, along with any supporting evidence.

2 In the schematic diagram of Figure 3D, it is stated that the extract induces c-JUN similarly to Hir/Ole. However, in the lower panel of Figure 3C, the c-JUN Western blotting data do not indicate statistical significance for the signal intensity upon extract treatment compared to the control. If the difference is statistically significant (p < 0.05), asterisks should be added to denote this.

Furthermore, there is no direct evidence demonstrating that the induction of BAK is c-JUN-dependent. Typically, c-JUN knockdown experiments followed by assessment of BAK induction (by RT-qPCR or Western blotting) are required to establish this dependency. If such confirmatory experiments have not been performed, the schematic presented in Figure 3D — which shows BAK induction by the extract alone and cleaved caspase-3 activation by Hir/Ole alone — implies that these are independent signaling events. In this case, we recommend revising the figure for clarity. Additionally, if c-JUN-dependent BAK induction has been reported in other published studies, the authors should include the appropriate citations.

Minor points

3 No description or mention of Figure 6C is provided in the text. Please check the manuscript and add the relevant explanation to the main text.

4 We noticed that the font size and style appear inconsistent for some words and sentences throughout the manuscript. For example, this issue is observed in lines 156–161 and for “H2O2” in line 570. We recommend that the authors carefully check the entire manuscript, including all other sections, to ensure consistent formatting.

Author Response

(Reviewer 1)

Major points

１. What is the difference between the non-fermented and fermented extracts? The authors have listed the molecules contained in the non-fermented extract in Supplementary Table 1; however, no information is provided regarding the composition of the fermented extract. It would be helpful to know how much the compositional profiles differ between the two extracts. If any experimental data on the components are available, please include them. If such data are not available, this point should be clearly discussed in the Discussion section. In particular, in the xenograft experiment shown in Fig. 5, the fermented extract appears to reduce tumor size more effectively than the bark extract. Is this difference statistically significant? If so, the authors should discuss the possibility that the fermented extract contains additional or different active components, along with any supporting evidence. In particular, in the xenograft experiment shown in Fig. 5, the fermented extract appears to reduce tumor size more effectively than the bark extract. Is this difference statistically significant? If so, the authors should discuss the possibility that the fermented extract contains additional or different active components, along with any supporting evidence.

[Answer]

These findings prompted us to further consider the potential differences between the non-fermented and fermented extracts, particularly in relation to their compositional profiles and biological effects. Although Supplementary Table 1 lists the shared components detected in the non-fermented extract, the unique composition of the fermented extract has not yet been fully characterized. Preliminary data suggest that fermentation alters the relative abundance of certain polyphenols, which may contribute to the observed differences in biological activity. In the xenograft experiments (Fig. 5), the fermented extract tended to reduce tumor size more effectively than the non-fermented extract. While this trend was reproducible, the statistical significance varied across experiments, indicating that additional or distinct active components may be generated during fermentation. Because the full compositional analysis of the fermented extract is ongoing in collaboration with the company, we acknowledge this limitation and emphasize that future studies are required to identify and validate the specific molecules responsible for the enhanced activity. These findings prompted us to further consider the potential differences between the non-fermented and fermented extracts, particularly in relation to their compositional profiles and biological effects.

Although Supplementary Table 1 lists the shared components detected in the non-fermented extract, the unique composition of the fermented extract has not yet been fully characterized. Preliminary data suggest that fermentation alters the relative abundance of certain polyphenols, which may contribute to the observed differences in biological activity.”
In the xenograft experiments (Fig. 5), the fermented extract tended to reduce tumor size more effectively than the non-fermented extract. While this trend was reproducible, the statistical significance varied across experiments, indicating that additional or distinct active components may be generated during fermentation.
Because the full compositional analysis of the fermented extract is ongoing in collaboration with the company, we acknowledge this limitation and emphasize that future studies are required to identify and validate the specific molecules responsible for the enhanced activity.

In the schematic diagram of Figure 3D, it is stated that the extract induces c-JUN similarly to Hir/Ole. However, in the lower panel of Figure 3C, the c-JUN Western blotting data do not indicate statistical significance for the signal intensity upon extract treatment compared to the control. If the difference is statistically significant (p < 0.05), asterisks should be added to denote this.

[Answer]

Thank you so much for your critical points. We have totally agreed on your criticism. We have done several experiments of western blotting using the new antibodies against BAK and c-JUN.

(i) The expression of c-Jun and BAK was examined by another several experiments. We confirmed the expressions of BAK and c-JUN were increased significantly. We have exchanged with new data.

(ii) We have done the experiments of shBAK and then examined the effects of c-Jun expression. As shown in the new data, we confirmed that the c-Jun was indeed affected by the shBAK treated rG2-DC-1C clones.

Minor points

3 No description or mention of Figure 6C is provided in the text. Please check the manuscript and add the relevant explanation to the main text.

[Answer]

Thank you so much for your kind suggestion. We have added the explanation of this result of Figure 6C in the main text.

[Answer]

As suggested by the reviewer, we have corrected the font size and style.

Author Response File: Author Response.docx

Reviewer 2 Report

Investigators compared unfermented and fermented Alnus japonica bark extracts with purified Hirsutanone (Hir) and Oregonin (Ore) (presumed to be from extracts) as anti-cancer agents using stem dcell enriched variant of HepG2 HCC. Results indicated extracts induce autophagy and Hir/Ore induce apoptosis. Data in Fig. 2 indicate increases in ROS. Fig. 3B indicates extracts increase p62 (suggesting autophagy inhibition). All groups inhibit invasion (Fig. 4) as well as tumors in vivo (Fig. 5). Chloroquine (also autophagy inhibitor) blocks extract effects (Fig. 6). Authors suggest extracts inhibit cancer stem cell features in HepG2 variant ans is more effective than pro-apoptosis Hir/Ore.

Overall, this manuscript was difficult to read and understand. Rationale is questionable and the overall conclusion is in doubt. Some suggestions are intended to perhaps guide authors to improve rationale and conclusion.

Abstract, last sentence of Results should be revised for clarity. Last sentence in conclusion also needs revision.
Introduction first paragraph can be shortened, too much detail if focus of paper is molecular mechanism.
Sentence in line 97 in Introduction should be removed or revised without stating "used as controls".
Introduction rationale is confusing. As I understand, comparing purified compounds Hir and Ore (found in Aj bark extracts) with whole bark extracts. Whole extracts induce autophagy whereas Hir/Ore induce apoptosis. Is one better than the other? Rationale of study needs better clarification.
Methods (and other areas) have some sentences crossed out which is inappropriate.
Methods 2.4 origins of recombinant lentivirus are not specified.
Methods 2.10 random check of primer sequences did not find correct matches using Blast search.
2.11 no references cited after "described previously ". Line 306-307.
Results 3.1 using extracts appear to show some anti-proliferation, unlike stated in text.
Fig. 1D use co-transfection with Jun/luciferase and Oct4 expression? Need to clarify in fig. legend and text.
Results are not making sense if increase in Oct4 also increases Jun yet RT-PCR shows extract decreases in Oct4 yet increases in Jun (1B). Are statistical analysis compared to control in 1C.?
Results 3.4 lines 628-633 needs clarification. Imply extract inhibits apoptosis?
Line 652-654 is incorrect.
Discussion line 797-799 is incorrect. Hir/Ore (pro-apoptosis) also have strong anti-cancer effect.
Discussion line 810-812, I also disagree.

Author Response

Reviewers 2:

Major comments:

Investigators compared unfermented and fermented Alnus japonica bark extracts with purified Hirsutanone (Hir) and Oregonin (Ore) (presumed to be from extracts) as anti-cancer agents using stem cell enriched variant of HepG2 HCC. Results indicated extracts induce autophagy and Hir/Ore induce apoptosis. Data in Fig. 2 indicate increases in ROS. Fig. 3B indicates extracts increase p62 (suggesting autophagy inhibition). All groups inhibit invasion (Fig. 4) as well as tumors in vivo (Fig. 5). Chloroquine (also autophagy inhibitor) blocks extract effects (Fig. 6). Authors suggest extracts inhibit cancer stem cell features in HepG2 variant and is more effective than pro-apoptosis Hir/Ore.

[Answer]

We sincerely thank you for your thorough and critical evaluation of our manuscript. Your comments have helped us recognize areas where our rationale and conclusions require clearer articulation. Below, we address your major concerns:

Overall rationale and conclusion Our study aimed to distinguish the biological effects of Alnus japonica bark extracts (both unfermented and fermented) from those of purified diarylheptanoids (Hirsutenone and Oregonin). While most prior reports emphasize apoptosis induced by Hir/Ore, our data reveal that the crude extracts predominantly activate BAK-dependent autophagy, which in turn suppresses cancer stem cell–like properties in the HepG2 variant. This distinction is important because the total biological activity of natural extracts cannot be fully explained by isolated compounds alone. The extracts represent a complex mixture, some components of which (e.g., flavonoids such as quercetin) contribute to autophagy induction. Thus, our rationale is to highlight that whole extracts exert broader, and in some contexts more effective, Cancer stem cells-like clone suppressive activity than purified pro-apoptotic components.

We acknowledge that our initial presentation may have obscured this rationale. In the revised manuscript, we will:

Shorten and sharpen the Introduction to focus on the mechanistic comparison (autophagy vs apoptosis).
Clarify that our conclusion is not that Hir/Ore lack anti-cancer activity, but rather that extracts act through a distinct autophagy-mediated pathway, which may complement apoptosis.
Revise the Abstract and Conclusion as well as Introduction sentences to ensure clarity and avoid overstatement.

Detailed comments:

[Aｔ Abstract, last sentence of Results should be revised for clarity. Last sentence in conclusion also needs revision.

[Answer]

Abstract and Conclusion: We will revise both for clarity and balance, emphasizing the mechanistic distinction without overstating superiority.

Introduction first paragraph can be shortened, too much detail if focus of paper is molecular mechanism.

[Answer]

Introduction: We will shorten the first paragraph, remove unnecessary detail, and clarify the rationale for comparing extracts vs purified compounds. I also shorten the introduction section to focus upon the molecular mechanism and remove the unrelated sentences.

Sentence in line 97 in Introduction should be removed or revised without stating "used as controls".

[Answer]

Line 97: We will remove the phrase “used as controls.”

Introduction rationale is confusing. As I understand, comparing purified compounds Hir and Ore (found in Aj bark extracts) with whole bark extracts. Whole extracts induce autophagy whereas Hir/Ore induce apoptosis. Is one better than the other? Rationale of study needs better clarification.

[Answer]

We appreciate this observation. Our rationale is to compare whole extracts (which induce autophagy) with purified Hir/Ore (which induce apoptosis). We will clarify that our goal is not to claim superiority but to highlight complementary mechanisms.

Methods (and other areas) have some sentences crossed out which is inappropriate.

[Answer]

As suggested by the reviewer, we have crossed out some sentences which was inappropriate in this Methods and other sections.

Methods 2.4 origins of recombinant lentivirus are not specified.

[Answer]

We have added the origins, the catalog numbers in the BRC Bank, in Japan, and the siRNA Center in Academia Sinica in Taiwan and their protocol information were noted.

Methods 2.10 random check of primer sequences did not find correct matches using Blast search.

[Answer]

As suggested by the reviewer, we made mistyped primer sequences. Thus, we have corrected the primer sequences.

The primer sequences used were as follows:

OCT4: forward 5′- GGGTTTTTGGGATTAAGTTCTTCA-3′, reverse 5′- GCCCCCACCCTTTGTGTT-3′
SOX2: forward 5′- GCTACAGCATGATGCAGGACCA-3′, reverse 5′- TCTGCGAGCTGGTCATGGAGTT-3′
KLF4: forward 5′-CATCTCAAGGCACACCTGCGAA-3′, reverse 5′- TCGGTCGCATTTTTGGCACTGG-3′
C-JUN: forward 5′-CCTTGAAAGCTCAGAACTCGGAG-3′, reverse 5′- TGTCTGCGTTAGCATGAGTTGGC-3′

Oct4 primer sequences was originate from our lab confirmed to recognize Oct 4 by using primer blast. KLF was confirmed same sequence as listed article published from oncogene. SOX2 and c-JUN shared the sequence as commercial products.

Oct4

OCT4-F	5’-GGG TTT TTG GGA TTA AGT TCT TCA-3’
OCT4-R	5’-GCC CCC ACC CTT TGT GTT-3’

KLF4　Tang, K., Yu, Y., Zhu, L. et al.Oncogene38, 6970–6984 (2019). https://doi.org/10.1038/s41388-019-0932-1
forward 5′-CATCTCAAGGCACACCTGCGAA-3′
reverse 5′-TCGGTCGCATTTTTGGCACTGG-3′

SOX2 Human qPCR Primer Pair (NM_003106)
forward: 5′-GCTACAGCATGATGCAGGACCA-3′
reverse: 5′-TCTGCGAGCTGGTCATGGAGTT-3′
c-Jun (JUN) Human qPCR Primer Pair (NM_002228)
forward 5′-CCTTGAAAGCTCAGAACTCGGAG-3′
reverse 5′-TGTCTGCGTTAGCATGAGTTGGC-3′

11 no references cited after "described previously ". Line 306-307.

[Answer]

We have added the correct reference [40] in the text.

Methods: We will correct inappropriate crossed-out sentences, specify lentivirus origins (2.4), provide accurate primer references (2.10–2.11), and cite missing references.

Results 3.1 using extracts appear to show some anti-proliferation, unlike stated in text.

[Answer]

We also measured cell viability using various cytotoxicity assays, including the trypan blue dye exclusion test, over an 8-day cultivation period (Figure 1A). After 8 days of culture, unfermented and fermented tree bark extracts decreased cytotoxicity by 22–30% ± 2.5% and 38–42% ± 3.5% in rG2-DC-1C cells, respectively. Treatment with fermented tree bark extracts did not show a significant reduction of cytotoxicity in rG2-DC-1C cells (40%) as compared with those of unfermented tree bark extract (20%) and Nir/Ore components (less than 5%). This is a meaning what we mentioned. We have corrected and deleted others.

1D use co-transfection with Jun/luciferase and Oct4 expression? Need to clarify in fig. legend and text.

[Answer]

In Fig.1D, we carried out co-transfection experiments using JUN-luciferase (WT and Mutant) constricts and expression vector EF-1 driven OCT4, which were described in the Materials and Methods section and Figure legends, as suggested by Reviewer 2.

Results are not making sense if increase in Oct4 also increases Jun yet RT-PCR shows extract decreases in Oct4 yet increases in Jun (1B). Are statistical analysis compared to control in 1C.?

[Answer]

Yes. I agree with your points. In rG2-DC-1C clones, the expression of OCT4 was decreased in the treatments with fermented or unfermented tree bark, but expression of C-Jun was increased in the cases of fermented or unfermented tree bark, in Hir/Ore treatment, although this clone was generated by the OCT4-c-Jun forced expression to show the positive feedback [30]. The treatments with fermented or unfermented tree bark reduced the OCT4 stemness character but increased the oncogenic character c-JUN based on the results of RT-PCR. However, if the expression of OCT as stemness marker was increased as the forced expression system, the cancer maker c-JUN promoter increased without any treatment, however, the fermented or unfermented tree bark exposure as induction of cancer character resulted in less sensitive activated the c-Jun promoter activity. Thus, these feedback-regulations between OCT4 and c-Jun were less sensitive than no treatment with the fermented or unfermented tree bark exposure (Figure 1C). Therefore, these feed-back control of cancer stemness (OCT4) and oncogenesis (c-JUN) in rG2-DC-1C clones was not functional when we treated these fermented or unfermented tree barks. We need more experiments to confirm these observations. We discussed this in Result section.

We also revised it from one more upper section to make sentences smoothly for the audiences.

Furthermore, we reported previously that the feedback loop between OCT4 and c‑JUN plays a critical role in maintaining stemness features of liver cancer stem cells [30]. To further examine this, we analyzed the transactivation of the c‑JUN promoter using a luciferase reporter assay with increasing doses of OCT4 (Fig. 1D). As expected, OCT4 induced activation of the c‑JUN promoter in the wild‑type rG2‑DC‑1C clone, whereas mutation of the OCT4 binding site (M4) abolished promoter activity.

However, treatment with fermented or unfermented bark extracts, as well as 20 μM Hir and 20 μM Ore, resulted in only modest and reduced induction of c‑JUN promoter activity compared with the untreated wild‑type control. These findings indicate that the feedback regulation between OCT4 and c‑JUN, which normally sustains stemness and oncogenic features in rG2‑DC‑1C clones, is attenuated under bark extract treatment. Notably, when OCT4 expression was elevated, the oncogenic activity of c‑JUN was repressed in the presence of fermented or unfermented bark extracts and Hir/Ore (Fig. 1C and D). We plan to investigate this feedback regulation further in future studies.

Results 3.4 lines 628-633 needs clarification. Imply extract inhibits apoptosis?

[Answer]

We appreciate your comment. Yes, as reported in the cited study, Alnus japonica hot water extract (AJHW) and oregonin inhibited apoptosis and muscle loss induced by H₂O₂ and dexamethasone. These effects were observed at relatively low concentrations, distinguishing them from other natural products that require higher doses. Thus, AJHW and oregonin were shown to attenuate apoptosis while promoting muscle protein synthesis and reducing degradation, supporting their potential relevance to sarcopenia.

Line 652-654 is incorrect.

[Answer]

We agreed on your criticism. Thus, we deleted this part to avoid the confusion.

Discussion line 797-799 is incorrect. Hir/Ore (pro-apoptosis) also have strong anti-cancer effect.

[Answer]

We agree that Hir/Ore also exert strong anti‑cancer effects via apoptosis. We will revise the Discussion to emphasize complementary mechanisms rather than exclusivity, correcting lines 797–799 and 810–812 accordingly.

Discussion line 810-812, I also disagree.

[Answer]

Total extracts---

Discussion: We will correct lines 652–654, 797–799, and 810–812 to acknowledge that Hir/Ore also exert strong anti-cancer effects via apoptosis, while extracts act through autophagy. Our revised discussion will emphasize complementary mechanisms rather than exclusivity.

Commitment to revision We appreciate your concern that the manuscript was difficult to follow. We will restructure the text for clarity, improve figure legends, and ensure consistency between results and interpretation. Our goal is to present a balanced, mechanistically sound comparison that highlights the translational potential of natural extracts as autophagy modulators, while recognizing the established apoptotic activity of Hir/Ore.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors (Kenly Wuputra and colleagues) have made their best efforts to investigate the molecular role of Natural Extracts of Alnus japonica in the anticancer activity against Liver Cancer Stem Cells and Tumorigenesis. I think, useful contribution for correction of certain oversights and small errors in the manuscript will be suitable for publication in the Antioxidants - MDPI journal. The authors should consider the following minor issues / suggestions to improve the strength of this manuscript:

The main concern of this manuscript is the requirement for the author to provide clear evidence regarding the biocompatibility of the extract, as this is essential for validating its potential biomedical applications.
The figure legend for Fig. 3 lacks essential methodological details. The authors should specify the number of independent experiments performed to ensure reproducibility and clarity of the results. Furthermore, the statistical methods used for data analysis, including the type of test and significance criteria, should be clearly stated in the legend or Materials and Methods section.
In Fig. 6C, the absence of error bars in the column representing Western blot quantification raises concerns regarding the presentation of data variability and reproducibility. Error bars are essential to indicate the degree of variation among replicates and to support the robustness of the findings. Additionally, the authors should explicitly describe the statistical methods used for data analysis, including the type of statistical test applied, the number of replicates considered, and the threshold for significance. This information should be included either in the figure legend or in the Materials and Methods section to ensure transparency and proper interpretation of the results.
Some of the blot images (such as,- Fig. 3A: ABCG2; Fig. 6C: BAK; etc.) are not sufficiently clear for proper interpretation. Therefore, I recommend that the authors repeat the corresponding experiments to ensure the reliability and clarity of the results presented.
Most of the subheadings in the Results section are not appropriate/incomplete; they appear to belong in the Materials and Methods section. For example, regarding the “Xenotransplantation Assay,” I would like to suggest that the authors provide additional details, including the purpose of the assay, the targeted compounds/drugs, and the cell type used.

None

Author Response

Reviewers 3

Major comments:

Authors (Kenly Wuputra and colleagues) have made their best efforts to investigate the molecular role of Natural Extracts of Alnus japonica in the anticancer activity against Liver Cancer Stem Cells and Tumorigenesis. I think, useful contribution for correction of certain oversights and small errors in the manuscript will be suitable for publication in the Antioxidants - MDPI journal. The authors should consider the following minor issues / suggestions to improve the strength of this manuscript:

[Answer]

Thank you so much for your kind questions and comments. We answered to your questions as below.

The main concern of this manuscript is the requirement for the author to provide clear evidence regarding the biocompatibility of the extract, as this is essential for validating its potential biomedical applications.

[Answer]

Thank you for raising this important point. We agree that biocompatibility is essential for validating the biomedical potential of Alnus japonica bark extracts. While our current study focused on anticancer activity in CSC‑driven hepatocellular carcinoma models, we recognize the need to assess safety in normal cells and tissues. In future work, we plan to perform viability assays in non‑cancerous hepatocytes, hemolysis tests, and in vivo toxicity studies including serum biochemistry and histopathology. These investigations will provide clear evidence of biocompatibility and strengthen the translational relevance of our findings.

The figure legend for Fig. 3 lacks essential methodological details. The authors should specify the number of independent experiments performed to ensure reproducibility and clarity of the results. Furthermore, the statistical methods used for data analysis, including the type of test and significance criteria, should be clearly stated in the legend or Materials and Methods section.

[Answer]

As suggested by the reviewer, we revised the Figure legends section and Materials and Methods in text of Figure 3. For Figure 3 B, more than three independent experiments were performed; statistical methods of one-way ANOVA were used with post hoc Tukey HSD compared between control group and each column treatments of specific protein. The significance shown in asterisk as * p<0.05, ** p<0.01, *** p<0.001 (n>3).

In Fig. 6C, the absence of error bars in the column representing Western blot quantification raises concerns regarding the presentation of data variability and reproducibility. Error bars are essential to indicate the degree of variation among replicates and to support the robustness of the findings. Additionally, the authors should explicitly describe the statistical methods used for data analysis, including the type of statistical test applied, the number of replicates considered, and the threshold for significance. This information should be included either in the figure legend or in the Materials and Methods section to ensure transparency and proper interpretation of the results.

[Answer]

We have repeated the experiments of Western blotting several times to confirm the results. WE added the information in Figure legends of Fig. 6 and Materials and Methods section in the text. For Figure 6 C the western blots were repeated for more than three independent experiments; statistical analysis was done by using two-way ANOVA followed with Bonferroni post hoc test. The significance of Control vs CQ 10 μM shown in asterisk as * p<0.05, ** p<0.01 (n>3).

Some of the blot images (such as,- Fig. 3A: ABCG2; Fig. 6C: BAK; etc.) are not sufficiently clear for proper interpretation. Therefore, I recommend that the authors repeat the corresponding experiments to ensure the reliability and clarity of the results presented.

[Answer]

As suggested by the reviewer, some indicated Western blotting especially BAK and BAX and cJUN were performed extensively to find the conclusive results. We thank reviewers for repeating the corresponding experiments, unfortunately due to the limitation of time, we were unable to repeat all recommended blots. However, we selected the most critical target BAK to ensure the significance of results presented. The repeated results were shown as the above answer, new statistical results were added to substitute the original Fig. 6 C.

Most of the subheadings in the Results section are not appropriate/incomplete; they appear to belong in the Materials and Methods section. For example, regarding the “Xenotransplantation Assay,” I would like to suggest that the authors provide additional details, including the purpose of the assay, the targeted compounds/drugs, and the cell type used.

[Answer]

As recommended by the reviewer, we have revised the subheadings in the Results section to ensure they are appropriate and complete. We trust that these changes will address Reviewer ’s concerns.

Results

3.1. Cellular Toxicity and Cancer Stemness Gene Profiles in rG2-DC-1C Cells Exposed to Fermented or Nonfermented Tree Bark Extracts During Prolonged Cultivation

3.2. Transcriptomic Characterization and Gene Clustering of Cell Growth, Cell Cycle, and Cell Death Pathways in Response to Fermented or Nonfermented Tree Bark Extracts

3.3. Comparative Analysis of ROS Activity in rG2-DC-1C Cells Treated with Fermented or Nonfermented Tree Bark Extracts

3.4. Expression of the AHR–NRF2 Axis and Cell Death-Associated Genes Following Treatment with Fermented or Nonfermented Tree Bark Extracts

3.5. Transwell Invasion Assay of rG2-DC-1C Cells Exposed to Fermented or Nonfermented Tree Bark Extracts

3.6. Xenotransplantation Assay Demonstrates Suppression of Tumor Formation in rG2-DC-1C Cells Treated with Fermented or Nonfermented Tree Bark Extracts

3.7. Identification of Autophagy-Inducing Components in A. japonica Bark Extracts

3.8. Effects of an Autophagy Inhibitor on Autophagy and BAK Expression Mediated by Fermented or Nonfermented Tree Bark Extracts

Reviewers 3

Major comments:

[Answer]

Thank you so much for your kind questions and comments. We answered to your questions as below.

The main concern of this manuscript is the requirement for the author to provide clear evidence regarding the biocompatibility of the extract, as this is essential for validating its potential biomedical applications.

[Answer]

The figure legend for Fig. 3 lacks essential methodological details. The authors should specify the number of independent experiments performed to ensure reproducibility and clarity of the results. Furthermore, the statistical methods used for data analysis, including the type of test and significance criteria, should be clearly stated in the legend or Materials and Methods section.

[Answer]

In Fig. 6C, the absence of error bars in the column representing Western blot quantification raises concerns regarding the presentation of data variability and reproducibility. Error bars are essential to indicate the degree of variation among replicates and to support the robustness of the findings. Additionally, the authors should explicitly describe the statistical methods used for data analysis, including the type of statistical test applied, the number of replicates considered, and the threshold for significance. This information should be included either in the figure legend or in the Materials and Methods section to ensure transparency and proper interpretation of the results.

[Answer]

Some of the blot images (such as,- Fig. 3A: ABCG2; Fig. 6C: BAK; etc.) are not sufficiently clear for proper interpretation. Therefore, I recommend that the authors repeat the corresponding experiments to ensure the reliability and clarity of the results presented.

[Answer]

Most of the subheadings in the Results section are not appropriate/incomplete; they appear to belong in the Materials and Methods section. For example, regarding the “Xenotransplantation Assay,” I would like to suggest that the authors provide additional details, including the purpose of the assay, the targeted compounds/drugs, and the cell type used.

[Answer]

As recommended by the reviewer, we have revised the subheadings in the Results section to ensure they are appropriate and complete. We trust that these changes will address Reviewer ’s concerns.

Results