2015/05/08 (Thu) MS submitted to Research Topic "Oral Oncology"
2015/05/30 (Sat) MS returned, interactive reviews activated:
Editor: Rui Amaral Mendes Catholic University of Portugal; Editor of J Carcinogenesis and Mutagenesis; Adhuant Prof at CWRU
2015/08/28 rejected... by Specialty Chief Editor JeanPascal Machiels
Reviewer#1:
加上morphology圖於Fig 1B
band quantitation
spheroid invasion
Reviewer#2:
移入IHC圖於Fig 5E
補上shRNA sequences and offtarget exp result 於Fig S1B
2015/07/13
Language:
Fine, but there are still numerous errors also in the amended sections.
E.g. Line 356: Interestingly
Line 379: only increment of DDR1 ???
Line 470: cancer
There are more! I just don't have the time to write them down all!
Use of a spell checker and proof-reading by a native English speaker is strongly recommended.
Objective errors:
Response to #1: The standardization of the cell culture medium could confound the experiment. The change of the cell culture medium to DMEM 10% FBS could alter transcriptional programs in cell lines that are normally grown in a different medium. The resulting PTK profiles are therefore flawed.
Response to #2: I still think that the choice of reference cDNA confounds the study. To identify a role in cancer biology, the expression profiles of normal tissue must be compared with that of cancerous tissue. That means a cDNA from normal keratinocytes should have been used as a reference. Comparing a mixed population of expression profiles from unrelated cancer cell lines with the profiles of OSCC cancer cell lines is like comparing apples with pears. The compared cell lines are of different tissue origin and may therefore express DDR1 at completely different levels. These comparisons do therefore not indicate a biological significance of the identified PTKs in oral cancer.
The authors comment below that EGFR expression is even higher in normal human keratinocytes and suggest that this may be caused by continuous EGF supplementation in the medium.
These statements clearly demonstrate that DDR1 could actually be strongly expressed in normal keratinocytes and is therefore not overexpressed in OSCC lines. Furthermore, this study demonstrates the limited applicability of cell line models, especially if grown under different conditions.
Response to #4: The HPV immortalised keratinocyte cell line shows indeed reduced DDR1 levels. However, a normal keratinocyte cell line such as HaCat or HOK is still not shown.
Response to #5: The explanation that DDR1 is the only overexpressed PTK in the analysed OSCC line is based on the assumption that the profiling results are correct. As I pointed out above (Response to #2), these data are flawed.
Response to #6: ANOVA analysis was performed but I'm surprised to see statiscial significance when the means are so close and the error bars overlap.
Introduction:
Lines 110-124: Good attempt but too long. No need to summarise all results.
M&M:
Response to #1: Satisfactory. As mentioned above, a table listing the characteristics of the used cell lines would have been clearer and more appropriate. (Lines 128-142: Content satisfactory. For clarity, a table would have been more suitable.
Response to #2: The technical issues have been sufficiently addressed. However, as outlined above, the profiling data are flawed due to use of an inappropriate control.
Response to 1: The standardization of the cell culture medium could confound the experiment. The change of the cell culture medium to DMEM 10% FBS could alter transcriptional programs in cell lines that are normally grown in a different medium. The resulting PTK profiles are therefore flawed.
I still think that the choice of reference cDNA confounds the study. To identify a role in cancer biology, the expression profiles of normal tissue must be compared with that of cancerous tissue. That means a cDNA from normal keratinocytes should have been used as a reference. Comparing a mixed population of expression profiles from unrelated cancer cell lines with the profiles of OSCC cancer cell lines is like comparing apples with pears. The compared cell lines are of different tissue origin and may therefore express DDR1 at completely different levels. These comparisons do therefore not indicate a biological significance of the identified PTKs in oral cancer.
The new evidence provided by the authors demonstrate that DDR1 could actually be strongly expressed in normal keratinocytes and is therefore not overexpressed in OSCC lines. I can also see no difference between normal controls and OSCC tissue (Fig. 5E). In summary, this study demonstrates the limited applicability of cell line models, especially if grown under different conditions.
Response to 2: The DDR1 levels are lower in the HPV-immortalised cell line but not in DOK. A different control keratinocyte cell line such as OKF6 or HOK should have been included.
Response to 3: has not been addressed by the authors
Response to 10 and 11: The authors do not describe the results of all tested cell lines (only for TW2.6). If there was no effect on cell migration in these cell lines , it should be explained!
Response to 15: Not adressed by the authors.
Response to 17: Unfortunately, the images are worse than the previous version. In the previous version, membranous staining for DDR1 was evident at least in the OSCC sample. What is presented here looks mostly cytoplasmic and there is a very strong background staining. I am concerned about immunostaining of this quality could be scored accurately. The staining pattern has also not been described in the results section.
I still think that the Discussion section lacks substance.
03/04/2014
Asia/Taipei
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PMID 24768818 Structural Mechanisms Determining Inhibition
1 - Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK
2 - Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02115, USA
3 - Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
4 - Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA
Correspondence to Nathanael S. Gray and Alex N. Bullock: N. S. Gray is to be contacted at: Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 250 Longwood Avenue, SGM 628, Boston, MA 02115, USA. A. N. Bullock. nathanael_gray@dfci.harvard.edu; alex.bullock@sgc.ox.ac.uk http://dx.doi.org/10.1016/j.jmb.2014.04.014
Edited by M. Guss
Abstract
The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer.
The discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among the receptor tyrosine kinases (RTKs) in being activated by interaction with the extracellular matrix [1,2]. Binding to triple-helical collagen is mediated by the receptor extracellular domains that include an N-terminal discoidin (DS) domain, a DS-like domain and a short juxtamem- brane (JM) region [3–5]. A single transmembrane helix links to the cytoplasmic domain, where a larger JM region precedes the catalytic C-terminal kinase domain. Both DDRs form constitutive dimers making them unusual among RTKs, which typically dimerize only upon activation [6–8]. DDRs regulate extracelular matrix remodeling, as well as cell adhesion, proliferation and migration [9]. DDR1 is expressed mainly in epithelial cells where it plays an important role in mammary gland development [10], whereas mesenchymal expression of DDR2 promotes bone growth, as suggested by dwarfism in DDR2 knock- out mice [11].
2015/06/20 a copy of Q and Response
1. There are no controls for Fig 1A and Fig S1 (Response: 補一張western於Fig S1, 並強調normal 或是 dysplastic 或是OSCC culture condition不同, 並且DDR1會因density不同表現量差異)
3. The E‐cadherin blot (Fig 2C) does not show a convincing reduction when DDR1 is knocked down. At the very least, this needs to be quantified. Similarly, the migration assay (Fig 4C) should be quantified. (Response: 玉蓮幫忙補quantitation data)
4. Experiments shown in Figs 3 and 4 use the pharmacological inhibitors imatinib and dasatinib. These are not specific for DDR1, yet no mention is made of inhibiting other targets, and effects on Abl or Src do not seem to have been considered. (Response: 補回上一個version那一段話)
5. In Fig 5A, treatment of cells with collagen is reported to increase DDR1 expression. Although the protein levels in HaCaT cells seem to increase, data for the other two cell lines are not convincing (densitometry would help) and it is unclear whether the changes seen in the QPCR experiment are significant (looks like around 20% to 50% increase). (Response:玉蓮幫忙補 Fig 5A quantitation data/ 查清楚Fig 5A lower panel是否有統計意義)
6. On p8, line 303, the authors mention that adjacent normal tissues were used. This is a concern, given the effects of field cancerization. (Response: WHT: 加回tissue microarry data直接比較 normal and OSCC)
The paper is well written, however there are some minor typographic and grammatical errors that should be corrected.
Examples:
‐ Current findings of the study or commonly known facts are often written in the past tense.
‐ "expressions" should be replaced with "expression levels", e.g. lines 305, 315, 338, 540, 607, etc
‐ Line 71: weakening
‐ line 245: Depending
‐ Line 255: associated
‐ Line 282: lesser extent etc (Response: thank you for your corrections, we have corrected all the errors accordingly)
Additional materials and methods found in figure legends instead.
Passages that belong into the Introduction section are found in the Results section. Passages that belong into the Results section are found in the Discussion section. The headings for each paragraph are not informative and should be rephrased. The descriptions should focus on what has been observed rather than speculative over‐ interpretations.
Overall, a very confusing presentation of the data. The information is all over the place and the reader has to search for the relevant information. The aims of the study have not been clearly formulated.
1. It is unclear why the cell culture medium was changed to a different medium one day before RNA extraction for RTK profiling. Since the culture conditions for the individual cell lines have not been described, it is not possible to evaluate if a change in culture medium could have unexpected effects on gene expression. (Response: culture media for individual cell lines are provided in revised Materials and Methods)
2. It is also not clear why a cDNA pool of multiple cancer cell lines (not specified from which tissues) was used as the control and as a baseline to determine DDR1 overexpression. This should have been done using cDNA from normal oral keratinocyte cell lines.(Response: the aim of PTK profiling is to identify OSCC-specific PTKs, compared to other malignancues, thus common, pooled cDNAs from common malgnancies was used)
3. The sequence of shRNA clones used for DDR1 knockdown has not been described. Furthermore, they have not been tested for off‐target effects. It is therefore impossible to know if the knockdown is specific for DDR1 or if other off‐target effects have contributed to the experimental outcome.(Response: sequences of shDDR1 have now been included; explain adjusted score > 1 in The RNA Consortium)
4. The DDR1 expression analysis has only been performed on OSCC cell lines but not on control normal keratinocytes. It is therefore not clear if DDR1 is overexpressed and to what degree.(Response: try to emphasize not comparable between normal and keratinocytes because cell density and culture component might affact)
5. The used drugs to inhibit DDR1 signalling are not specific for DDR1 but also effect on other RTKs (see literature). Therefore, the conclusions from the experiments need to be reevaluated and more carefully phrased.(Response: list of imatinib-sensitive and dasatinib-sensitive tyrosine kinases)
6. The correlation between DDR1 expression and oral tumor stage should have been statistically analysed using ANOVA and not by multiple t‐tests.(Response: ANOVA has been used to evaluate the correlation between DDR1 expression and pathological staging as described in Methods, Results and Legend of Fig 5C)
The experiments have been performed to a good standard and the figures generally are of good quality. However, some methodological errors could confound the results of the study and the interpretation of the data is occasionally incorrect or too speculative (see comments in individual sections). (Response: please see individual responses in the following)
The title only refers to the overexpression aspect of DDR1 but does not refer to the other findings of the study or, more importantly, does not send a key message, for example what the role of DDR1 is. (Response: too bad, we don't have a clear answer yet)
The introduction starts well but is too short and misses some information which can occasionally be found in other sections of the manuscript. The following aspects should be included or moved into the introduction from other sections of the manuscript:
‐ The roles of TGFB1 and collagen in oral submucous fibrosis or oral cancer should be made clearer (more carefully phrased). The cited studies merely show correlations and do not prove a causal effect of TGFB1 on collagen expression. (cite Khan Plos one 那一篇 23284772; )
‐ A summary of DDR1 isoforms including Protein domains and their known functions (see lines 201‐209 in Results section)
‐ The used cell lines have been published and a reference to their basic charcteristics could be made.
‐ Lines 271‐279 of results section belongs into Introduction.
‐ The concluding paragraph is incomplete, too speculative and somehow disconnected. The purpose of the study could be made clearer.
1. Not all used cell lines have been described in the M&M section. For example, A431 is missing. Furthermore, the basic characteristics of the used cell lines as well as the individual culture conditions could be briefly summarised rather than just referencing previous papers. It does not help with the interpretation of the results if the material is not described properly. For exampl,e in lines 364‐365 of the Discussion section, the authors mention that three of the analysed cell lines have very different characteristics but they do not elaborate on this and just refer to unpublished results. It is intriguing, for example, that the OC3 cell line shows a mesenchymal cell morphology (non‐keratinocyte) which is interesting considering that this cell line has also very different molecular characteristics. (Response:
2. The PTK profiling could benefit from more details, for example how qPCR data was analysed (for example this information can be found in the figure legend of Table1!) and why the baseline for comparison was a pooled cDNA sample from a range of different cancer cell lines and not from normal keratinocytes (e.g. HOK which have been used for other experiments in this study). The qPCR primers and their properties should be published as supplementary material in order to allow evaluation if these comply with accepted standards for qPCR primer design. For example, using the delta‐delta Ct method with SYBR Green chemistry is questionable unless the primers have been rigorously tested and validated. (Response: patent-related concern)
3. The target sequences for the shRNA constructs need to be made available and there needs to be a comment on specificty and off‐target effects. Could go in Supplement. (Response: TRC sequences will be provided)
4. The substances in which the PTK Inhibitors were dissolved should be mentioned. (Response: vehecial controls??)
The statistical analysis of DDR1 expression with different tumor stages should have been peformed using ANOVA and not multiple t‐tests. (Response: ANOVA has been used to evaluate the correlation between DDR1 expression and pathological staging as described in Methods, Results and Legend of Fig 5C)
1. It is not clear what the criteria were for stating that the mentioned 5 PTKs were overexpressed. The cDNA control from a pool of various cancer cell lines (what tissues?) is not a good baseline since any of these PTKs might be overexpressed in some of these cancers but not in others. Biological significance can be better evaluated by comparing with normal keratinocytes (see point 2.) (Response: 不不不, 是故意要比cancer cell lines的啊, 要找出OSCC specific or PTKs preferentially expressed in
2.As already mentioned, the western blots showing DDR1 expression should include a normal keratinocyte control to allow the conclusion of DDR1 overexpression (Fig 1 & S1 & S2). a low and high expressing control could ideally be included. (Response:
3. The title "Overexpression of DDR1 contributes to cell growth of oral cancer cells" is misleading and should be changed to "Knockdown of DDR1 induces growth arrest and apoptosis of oral cancer cells".
The argument for oncogene addicition of the studied OSCC cell lines is speculative since knockdown of DDR1 has not been performed in normal oral keratinocytes. For example, if these cells also undergo apoptosis, it can be concluded that DDR1 is simply an important protein for survival of oral keratinocytes. To investigate oncogenic activity, DDR1 should be overexpressed in normal oral keratinocytes. (Response: constitutively active vs collagen-stimulated active)
4. Line 240: "autophosphorylated" ??? or do the authors mean "constitutively phosphorylated"? (Response:
5. The results from the analysis of potential downstream targets of DDR1 signalling (Fig 2c) should be rpoperly explained and discussed. Potential mechanism? (Response:
6. The study concluded that the effect of PTK inhibitory drugs on OSCC cell survival was dosage‐dependent. However,only very high doses have a differential effect in OSCC lines compared to controls. Are these doses physiologically relevant or feasible for therapy? (Response:
7. The authors claim that "among the four OSCC cell lines, OC3 had the lowest amount of DDR1 and was less responsive to inhibitor treatments, suggesting that drug sensitivity is related to the DDR1 expression level."However, OC3 is sensitive to dasatinib (Fig 3D), and considering the known unspecificity of both PTK inhibitors, this conclusion is not valid. The different behaviour of OC3 cells could be explained by different cellular characteristics of this cell line such as acquirement of different cell fate. This hypothesis is supported by the authors claims that the OC3 cell line has a mesenchymal cell morphology and is also supported by the different molecular data shown in Figure 2c. (Response:
8. Line 263: refer to Figure 3a,b ? (Response:
9. Line 267: Imatinib and dasatinib are not specific "DDR1 Inhibitors" (Response:
10. "DDR1 is involved in collective invasion of OSCC TW2.6 cells." It should be noted in this paragraph that the other cell lines were also tested but did not exhibit cell cohesiveness. this is only mentioned in the Discussion section but a more comprehensive description would help the reader in understanding why only one cell line has been analysed. (Response:
11. OEC‐M1 cell cohsion data mentioned in text but not shown in Figure 4. (Response:
12. Line 284: replace "actomyosin activity" with "myosin expression". Is presence of myosin between cells generally accepted as an indicator of loss of cell cohesion? (Response:
13. Lines 289‐290: A cadherin immunostaining experiment would clarify this point. Why has it not been performed?
14. Results and Fig. 5a Collagen stimulation in oral keratinocytes results in minimal increase in DDR1 Expression. is this statistically significant? Why not? (Response:
15. Line 306+307: How can it be explained that only Col4a6 is co‐overexpressed with DDR1 when all collagens seem to stimulate DDR1 expression/activity in normal cells whereas DDR1 activity is independent of collagen stimulation in OSCC cells? What does that mean biologically? (Response:
16. Are the DDR1 expression values shown in Figures 5c and 5d from the microarray or from validated qPCR data? Fig 5c: Data needs to be statisticall analysed using ANOVA. (Response: ANOVA has been used to evaluate the correlation between DDR1 expression and pathological staging as described in Methods, Results and Legend of Fig 5C)
17. Unfortunately, the most exciting part of the study, the immunohistochemical analysis of collagen and DDR1 expression in oral tumours, has been buried as supplementary data (Supplementary figure S3) and has been insufficiently described in the Material and Methods section and the Results section. The images in Figure S3 are displayed at too high magnification yet too small to recognise details and therefore need reworking. Both the epithelial and mesenchymal (stromal) cell compartments should be visible and the images should have the same oientation. The origin of the analysed tissues should be described as well as any other information (e.g. patient data?). It needs to be made clear that increased expression levels of DDR1 positively correlate with tumor grade (if correct!). The source of collagen antibodies needs to be mentioned.
I would suggest to include the immunohistochemical analysis in the main manuscript and move other, less important aspects into the supplement (if required). (Response:
Notwithstanding the concerns mentioned above, the experiments have been performed well. (被您批評得一無是處了嗚嗚) However, the lack of cohesion is evident. The study would benefit from focused experiments that tie all the experiments together into an interesting story. Focus should be on biological or clinical relevance and conclusions should be
less speculative. (Response:
The aims of the study have not been clearly formulated, however the Discussion makes a good attempt. (Response:
The Discussion is very short and lacks substance. It is mainly a summary of the Results and Introduction sections. Comparison with DDR1 function in other contexts is sparse. Biological/clinical relevance or mechanistic insights have not been discussed. The links to oral cancer are insufficiently discussed. (Response:
See examples above.
The conclusions are frequently either too far‐fetched and speculative or absent. Relevant experiments could be performed to fill the gaps in knowledge.
Overall, there are still too many gaps in this study to conclude that DDR1 could be a therapeutic target in oral cancer. (Response:
2015/02/18 懺悔記
Frontiers in oncology
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DDR1 Inhibitors相關
J Mol Biol. 2014 Jun 26;426(13):2457-70. The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and alphaD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a beta-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.
代Maruko移轉NAS上資料
老闆我後來挑中的是:
DDR1 MS Figures回應
Referee #1的所有concern
1. Methods and reagents are poorly described.
–Figure 4. These data are not well described and therefore unconvincing.
#The conclusion that "in addition to the kinase activity, other structural domains of DDR1 may also contribute to cell fate determination" has not data and mechanistic bases and thus seems to be a total speculation, especially using non-specific inhibitors. Thus, the data and conclusions of Figure 6 are questionable.
–Page 12: "Thus, DDR1 is likely to be the final effecter of the TGFB1-collagen accumulation axis, which transmits and executes oncogenic processes in oral epithelial cells." This claim is over-reaching as there is no experimental proof to back it up.
–Supplement Figure 1. How are DDR1a and DDR1b differentiated in the western blot from the other isoforms when the antibody recognizes all isoforms?
Referee #2 的所有concern
Major concerns:
1. The authors presented too many unsorted data especially lots of western blots throughout the manuscript, but did not provide interpretations about those data, how they can support the main aims. For example: many blots showed expression of cell cycle regulators in OSCC cells transfected with control-shRNA or DDR1-shRNA, but the authors did not provide mention why DDR1 depletion induced expression of some cell cycle regulators but reduced expression of the similar cell cycle regulators. There are many overstatements and over-interpretation throughout the manuscript.
2. The authors did not provide direct evidence on that imatinib or dasatinib caused severe cell growth arrest was associated with its inhibition of DDR1. Unless they show that imatinib or dasatinib directly inhibit DDR1 phosphorylation kinetics.
3. The authors state that DDR1 overexpression is associated with pathogenesis of OSCC, but they did not introduce overexpression of DDR1 into OSCC cells. The overexpression experiments need to be performed to support their statement about the role of DDR1.
4. The conclusion on that (page 4-5) "overexpression of DDR1 is also a hallmark of oral cancer cells" is overstated. They only analyzed 40 patients by immunohistochemical analysis and observed that expression levels of DDR1 expression and collagens were elevated in cancer specimens compared with normal tissues. No functional study using cell line systems to examine whether overexpression of DDR1 may increase tumor cell growth and survival.
5. Materials and methods section, experimental procedures need to be described in great details. No description about how cell cycle analysis is performed. How cell growth in Figure 1 is determined. (OK now)
6. Figure 1. Figure 1b, It is not clear how cell growth is determined. Stringent methods using flow cytometry analysis to determine viable cells are required.
7. Figure 2a. It is not appropriate to use trypin-blue to measure the rate of apoptosis. Statistical calculations need to be indicated. (OK now)
8. Figure 3a. It is difficult to interpreted Figure 3a. The authors stated that Four OSCC cell lines were treated with collagen 1 for 18 hours to increase DDR1 expression. The authors did not explain how this experiment is performed. The authors did not use specific antibody against tyrosin-phosphorylated DDR1. It is not appropriate to use word "overexpressed DDR1" to describe that DDR1 expression is increased after treatment with collagen 1. It is difficult to understand the conclusion from the authors: "overexpressed DDR1 in OSCC cells was constitutively active, regardless of collagen stimulation". The quality of bands is poor. Semi-quantification of bands of western blots in Figure 3 needs to be performed to show whether increase in DDR1 expression is significant in these cell lines.
Figure 4b.
I am surprised that DDR1 shRNA depletion reduced CDK1 expression, but increased CDK2, cyclin D1 expression which are the key cell cycle regulators to promote cell proliferation. This indicates that DDR1 depletion may have positive effect on cell growth. To clarify this issue, the authors need to perform Brdu-based proliferation assay using cells transfected with Sh-control RNA and ShRNA-DDR1. These data are discrepancy to the data showing that DDR1 depletion reduced cell growth and increased apoptosis in these cancer cell lines.
Figure 5. It is not appropriate to state that Imatinib and dasatinib are DDR1 inhibitor. The only data which is relevant to the statement was that treatment of cells with Imatinib or dasatinib inhibited phosphor-tyr expression in DDR1-associated immunocomplex. It is not known whether these two kinase inhibitors indeed inhibited DDR1 phosphorylation or expression. The authors did not provide sufficient evidence to support their statement. The quality of Figure 5C is poor. The result from Figure 5C is inclusive. Why the authors did not perform same experiments using cells which have been treated for 24 or 48 hours to examine both P-Tyr and expression of DDR1.
Figure 6a and b. The quality of upper panels of Figure 6a and b are poor. The statistical significance from three independent experiments should be indicated in Figure 6a and b. (OK now)
CDDis的二位Reviewer之critiques