Amorphigenin from Amorpha fruticosa L. Root Extract Induces Autophagy-Mediated Melanosome Degradation in mTOR-Independent- and AMPK-Dependent Manner
and Kwang Dong Kim 1,2,4,*Round 1
Reviewer 1 Report
The authors present a study that shows the effect of an extract of the root of the herb Amorpha fruticosa L. on depigmentation in B16F10 mouse cells. In addition, they identify an active ingredient in these extracts as the inducer of this depigmentation.
The authors also show that the depigmentation in B16F10 mouse cells is due to the induction of autophagy by the active agent they identify as amorphigenin, and that AMP-activated protein kinase (AMPK) is involved in this process.
From my point of view, the paper qualifies for publication in this journal once a small editorial error has been corrected.
Line 352. "fpcused" should read "focused".
Author Response
Comment 1. From my point of view, the paper qualifies for publication in this journal once a small editorial error has been corrected. Line 352. "fpcused" should read "focused".
Answer) Thank you for your kindness. We correct “fpcused’ to ‘focused’.
Reviewer 2 Report
In their present manuscript, Ki Won Lee and colleagues report a role for amorphigenin, a natural compound derived from A. fruticose root extracts, as a depigmentation inducer in mouse B16F10 melanoma cells. The authors describe the effect to result from AMPK-induced autophagy. As a consequence, inhibiting autophagy or blocking lysosomal protein degradation either via selected pharmacological inhibitors or via siRNA-mediated knockdown or knocking down AMPK itself, rescues melanosome formation, protein levels of pigmentation associated genes, and pigmentation. Overall, this is a very nice study with impressively clear data. Experiments are well explained and conservatively interpreted. I have only a few very minor suggestions:
1) Line 36: A space is missing within “processthat”.
2) Line 188: It is stated on line 188 that Kojic acid was used as a positive control, but the exact same statement was already made (and giving more detail) two sentences earlier on line 185.
3) Line 197: The line contains an additional space, which can be deleted.
4) Line 225: The statement “The dot formation of Trp-1 …” sounds somewhat suboptimal in English. Perhaps a rephrasing as “The Trp-1-associated dotty subcellular pattern, representing melanosomes, …”.
5) In figure 2, panel “F” is incorrectly labeled as “D”.
6) Line 342: It is stated that “Rapamycin, a representative non-selective autophagy inducer of autophagy and an mTOR inhibitor, successfully induces autophagy through mTOR inhibition and AMPK activation but did not induce depigmentation in α-MSH-stimulated B16F10 cells”. In this sentence, the word autophagy is duplicated (either say “autophagy inducer” or “inducer of autophagy”, not both). But more importantly, unless I have missed it, the study does not actually show that rapamycin “successfully” induces autophagy in B16F10 cells. It only shows that rapamycin inhibits mTOR (Fig. 5A) and does not induce depigmentation (Fig. 5B). Moreover, contrary to above statement, rapamycin does not activate AMPK in B16F10 cells (Fig. 5A), as judged by the absence of a phospho-AMPK band in the rapamycin condition.
7) I am not entirely sure which PMEL antibody was used for the experiments, but the size labeling of “15 kDa” on the Western blots does not fit well with any known PMEL fragments (the C-terminal Mb fragment and the fibril-associated RPT fragments all typically run larger than 25 kDa). I recommend the authors re-check the size labeling on the Wester blots and mention the name of the used antibody in the Materials & Methods section.
Author Response
Comment 1. Line 36: A space is missing within “processthat”.
Answer) We edited “processthat” to “process that”.
Comment 2. Line 188: It is stated on line 188 that Kojic acid was used as a positive control, but the exact same statement was already made (and giving more detail) two sentences earlier on line 185.
Answer) Thank you for your kindness. According to your advice, we deleted the sentence “Kojic acid was used as a positive control of depigmentation agent.” on line 188.
Comment 3. 3) Line 197: The line contains an additional space, which can be deleted.
Answer) We deleted the additional space on line 197.
Comment 4. 4) Line 225: The statement “The dot formation of Trp-1 …” sounds somewhat suboptimal in English. Perhaps a rephrasing as “The Trp-1-associated dotty subcellular pattern, representing melanosomes, …”.
Answer) Thank you for your kindness and a good point. The phrase was replaced with “The Trp-1-associated dotty subcellular pattern, representing melanosome,” as you suggested.
Comment 5. 5) In figure 2, panel “F” is incorrectly labeled as “D”.
Answer) We replaced the second “D” with “F” in figure 2.
6) Line 342: It is stated that “Rapamycin, a representative non-selective autophagy inducer of autophagy and an mTOR inhibitor, successfully induces autophagy through mTOR inhibition and AMPK activation but did not induce depigmentation in α-MSH-stimulated B16F10 cells”. In this sentence, the word autophagy is duplicated (either say “autophagy inducer” or “inducer of autophagy”, not both). But more importantly, unless I have missed it, the study does not actually show that rapamycin “successfully” induces autophagy in B16F10 cells. It only shows that rapamycin inhibits mTOR (Fig. 5A) and does not induce depigmentation (Fig. 5B). Moreover, contrary to above statement, rapamycin does not activate AMPK in B16F10 cells (Fig. 5A), as judged by the absence of a phospho-AMPK band in the rapamycin condition.
Answer) Thank you for your good points. As your advice, we modified “non-selective autophagy inducer of autophagy” to “non-selective autophagy inducer”. And as you said, we did not show that rapamycin induces autophagy and phospho-AMPK activation. Therefore, we deleted the sentences “successfully induces autophagy through mTOR inhibition and AMPK activation”
7) I am not entirely sure which PMEL antibody was used for the experiments, but the size labeling of “15 kDa” on the Western blots does not fit well with any known PMEL fragments (the C-terminal Mb fragment and the fibril-associated RPT fragments all typically run larger than 25 kDa). I recommend the authors re-check the size labeling on the Wester blots and mention the name of the used antibody in the Materials & Methods section.
Answer) Thank you for your exact point. The antibody we used can bind with 1-100 amino acids of PMEL protein. Thus, this antibody can detect PMEL fragments of Mα, MαN, and NTF. In our manuscripts, we showed the NTF of mouse PMEL fragments which 17 kDa of molecular weight approximately. And as you mentioned, we stated the antibody name in the Materials & Methods section.
