Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases. Arabidopsis thaliana (hereafter Arabidopsis) leucine-rich repeat receptor kinases (LRR-RKs) FLS2 and EFR recognize the bacterial PAMPs flagellin (or its derived epitope flg22) and elongation factor-Tu (or its derived epitope elf18), respectively  and receptor proteins (RPs) are the main ligand-binding cell-surface receptors perceiving self, non-self, and modified-self molecules . For exaectively . Both FLectively .Catharanthus roseus RECEPTOR-LIKE PROTEIN KINASE 1-LIKE (CrRLK1L) FERONIA (FER) and the GPI-anchored protein LORELEI-LIKE GPI-ANCHORED PROTEIN 1 (LLG1) are required for flg22-induced FLS2-BAK1 complex formation  and single-particle tracking to analyze the lateral mobility of FLS2-GFP in transgenic  reports , we obse reports . Consist reports . Compara reports , showed an in WT . To anal mutants . To subs mutants . As expean in WT , indicatMedicago truncatula and yeast, alteration of nanodomain localization has been linked to impaired protein accumulation at the plasma membrane due to increased protein endocytosis  shows stunted growth and salt hypersensitivity phenotypes reminiscent of fer-4 , previously used to assess protein complex formation  in the fer-4 mutant background  C-terminally fused to GFP, expressed in fer knock-out backgrounds  was used as WT control. The fer-4, fer-4/pFER::FER-GFP  medium supplemented with vitamins, 1% sucrose and 0.8% agar.:FER-GFP , fer-4/pERKD-GFP , fer-4/pMalA-GFP , Col-0/pMalA-GFP , Col-0/pFLS2-GFP , lrx3/4/4ΔE-FLAG  lines we4ΔE-FLAG  was cros-mCherry  was crosenerated . DespiteThe flg22 (QRLSTGSRINSAKDDAAGLQIA), elf18 (SKEKFERTKPHVNVGTIG), and RALF23 (ATTKYISYGALRRNTVPCSRRGASYYNCRRGAQANPYSRGCSAITRCRR) peptides were synthesized by EZBiolab (USA) with a purity of >95%. All peptides were dissolved in sterile purified water.ROS burst measurements were performed as previously documented . At leasfer-4, lrx3/4/5, and 35S::LRR4-Cit seedlings (n = 9–12) were transferred for additional 3 days to 3 mL liquid ½ MS medium containing different concentrations  of flg22 or the appropriate amount of solvent. The seedlings were then placed on solid MS plates before scanning. Root length was measured using ImageJ.Three-day-old Col-0, For confocal microscopy and VA-TIRF microscopy experiments, surface-sterilized seeds were individually placed in line on square Petri dishes containing 1/2 MS 1% sucrose, 0.8% phytoagar, stratified 2 days in the dark at 4°C, then placed in a growth chamber at 22°C and a 16 hr photoperiod for 5 days. Seedlings were mounted between a glass slide and a coverslip in liquid 1/2 MS, 1% sucrose medium. For VA-TIRF microscopy experiments, 2–4 seedlings were sequentially imaged for each genotype and/or condition. To test the effect of RALF23 on FLS2-GFP dynamics and nanodomain organization, seedlings were preincubated in 2 mM MES-KOH pH 5.8 for 3–4 hr prior treatment. Seedlings were imaged 2–30 min after treatment.Confocal microscopy was performed using a Leica SP5 CLSM system  equipped with Argon, DPSS, He-Ne lasers, hybrid detectors, and using a 63 × 1.2 NA oil immersion objective. GFP was excited using 488 nm argon laser, and emission wavelengths were collected between 495 and 550 nm. mCherry was excited using 561 nm He/Ne laser, and emission wavelengths were collected between 570 and 640 nm. Propidium iodide was imaged using 488 nm and 500–550 nm excitation and emission wavelengths, respectively. In order to obtain quantitative data, experiments were performed using strictly identical confocal acquisition parameters , with detector settings optimized for low background and no pixel saturation. Pseudo-color images were obtained using look-up-table (LUT) of Fiji software .TIRF microscopy was performed using an inverted Leica GSD equipped with a ×160 objective , and an Andor iXon Ultra 897 EMCCD camera. Images were acquired by illuminating samples with a 488 nm solid-state diode laser set at 15 mW using a cube filter with an excitation filter 488/10 and an emission filter 535/50 for FLS2-GFP and FER-GFP. Optimum critical angle was determined as giving the best signal-to-noise for our sample and was kept fixed for each experiment. Images time series were recorded at 20 frames per second (50 ms exposure time) for 0)2 + (y – y0)2 and D = MSD/4t, where x0 and y0 are the initial coordinates, and x and y are the coordinates at any given time, and t is the time frame.To analyze single-particle tracking experiments, we used the plugin TrackMate 2.7.4  in Fiji Genotype and/or treatment-dependent variation in fluorescence intensity of FLS2-GFP and fluorescence pattern of FLS2-GFP and BAK1-mCherry compromised the use of a unique set of parameters to compute nanodomain size and density across the different experiments. To uniformly quantify differences in membrane organization of both FLS2 and BAK1 across all experiments, we used the SCI that was shown to be largely insensitive to variation in fluorescence intensity . Quantif2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4, 1 mM phenylmethanesulfonyl fluoride, and 0.5% IGEPAL. For immunoprecipitations, α-rabbit Trueblot agarose beads (eBioscience) coupled with α-FLS2 antibodies  and/or RALF23  were added and incubated for 10 min. Seedlings were then frozen in liquid N2 and subjected to protein extraction. To analyze FLS2-BAK1 receptor complex formation, proteins were isolated in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, 1% protease inhibitor cocktail (Sigma-Aldrich), 2 mM Natibodies  or GFP-TProtein samples were separated in 10% bisacrylamide gels at 150 V for approximately 2 hr and transferred into activated PVDF membranes at 100 V for 90 min. Immunoblotting was performed with antibodies diluted in blocking solution (5% fat-free milk in TBS with 0.1% [v/v] Tween-20). Antibodies used in this study were α-BAK1 1:5000; , α-FLS2 t-test Mann–Whitney test.Statistical analyses were carried out using Prism 6.0 software (GraphPad). As mentioned in the figure legends, statistical significances were assessed using nonparametric Kruskal–Wallis bilateral tests combined with post-hoc Dunn’s multiple pairwise comparisons or using a two-way nonparametric Student’s FER (AT3G51550), LRX3 (AT4G13340), LRX4 (AT3G24480), LRX5 (AT4G18670), RALF23 (AT3G16570), FLS2 (AT5G46330), BAK1 (AT4G33430). In elegant quantitative live-cell imaging and biochemical experiments, the authors show how activity of the plant immune signaling complex FLS2-BAK1 is affected by nanoscale mobility behaviors mediated through peptide signaling and the receptor kinase FERONIA (FER). Additionally, they are able to define separable roles for FER domains in different biological activities. The details of this work advance our understanding of plant immunity, but also provide generalizable concepts about the roles of nanoscale organization in signaling. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Jan Petrášek (Reviewer #3).Thank you for submitting your article "Regulation of immune receptor kinases plasma membrane nanoscale landscape by a plant peptide hormone and its receptors" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:From the written reviews and discussion, there was much enthusiasm for the work, but two general concerns emerged: (1) replicated data were not fully analyzed and reported and (2) the ability of a broad audience to engage with and appreciate the detailed work would be enhanced by modifications to the text. From the individual reviews appended below you will find specific examples of places where modifying text or presentation would have the largest impact.1) That "similar results were found among three replicates" is mentioned in nearly every figure, but these replicates are not shown. After a healthy discussion among the reviewers about what was essential to show, all three reviewers concurred that in the cases of single particle tracking and the quantitative imaging of mobility , the data from the three replicates should be analyzed and the differences between experiments reported. This is in part because this paper has the potential to serve as a standard bearer for this technique.In the case of Western blots, showing a single representative blot was fine, but the band intensities should be quantified using standard densitometry scans. We also encourage the authors to include the replicate, uncropped, blots in a data supplement. For the confocal images of leaves in Figure 3C-D, the single set of images is also fine, but additional details about what constitutes a replicate (see comment from Rev 1) is needed. For other phenotypic measurements like seedling fresh weight, reporting the single replicate is fine.2) A weak experimental point is the examination of protein accumulation on the plasma membrane – which is used to show that the nanodomain results in the stabilization of FLS on the membrane, preventing them from being endocytosed. Here single channel images are used to determine the amount of protein based on fluorescent intensity without further controls . Additionally, it is not shown whether fer mutants have altered endocytosis in general. Please either include supporting experiments or modify the text to indicate the limitations of the current study.3) The manuscript is very detailed as it described the nanoscale localization of receptor kinases, and the jargon and extensive use of abbreviations makes it challenging for readers outside the plant immune signaling world. Additional discussion and presentation of these findings in an integrative model would help to make the details illustrative of general signaling rules and would broaden the impact of this work. In addition, the phrasing of the abstract that includes mention of a "plasma membrane nanoscale landscape" may lead one to expect that paper will focus on the "landscape" of the PM and the structural changes in the PM provoked by the activity of individual receptor kinases. However, the main focus in on the complex dynamics of individual receptor protein kinases with the PM – no less interesting, but not completely aligned with the abstract.4) Many of the experimental details or choices are not fully explained; for example, FER effects on cell wall integrity are mostly known from root hair work, but the experiments here focus on cotyledon epidermal cells. Please add some rationale for experimental design choices, be clear about what a "sample" or "replicate" entails, and mention potential limitations of the current work.Reviewer #1:How does a protein identified as having a role in many different and seemingly independent plant responses act in specific ways in those responses? An example in plants is the receptor kinase FERONIA (FER) that has been implicated in numerous processes including cell wall integrity signaling and response to biotic and abiotic stresses. Using a series of cell biological and biochemical approaches, the authors show, using the intensively studied FLS2-BAK1 immune receptor complex and its previous identified ligands and signaling potentiators or inhibitors, that different domains and activities of FER have discrete roles in different cellular events. They further show that FER and LRX proteins may affect the mobility of plasma membrane proteins in their nanodomains, though precisely how loss of FER or LRX leads to changes in nanodomain properties is not entirely clear. This detailed work adds interesting and important information about how individual proteins and cellular milieus can alter signaling. Additional discussion and presentation of these findings is an integrative model would help to make the details illustrative of general signaling rules and would broaden the impact of this work outside of the plant immunity niche.From the set-up of this paper, I imagined that the authors would show a unified way in which FER and/or LRXs affected a cellular feature (e.g. nanodomain formation) and this would account for multiple activities. The results of their experiments, however, showed that they can separate activities of FER . I am left not quite knowing how direct FER activity is on FLS2-BAK1 behaviors. I was expecting it to be indirect through some nanodomain organization, but I failed to find this thread followed through. The direct effects of FER are also interesting results, but as an outsider to the PTI and cell wall integrity fields, I find it challenging to synthesize the results into a general overall model. I don't think the authors need more experiments, but I do think a figure that summarizes their updated concept of how FER works would make this paper accessible and appreciated by a wider audience.Reviewer #2:This paper provides insight into the molecular mechanisms which mediate a plants immune response. Specifically looking at the dynamic organization of nano-domains of immune receptors, where the role of hormone peptides, co-receptors and the cell wall upon this domain are examined. This thus provides insights into overall plant physiology and into the finer details of membrane dynamic organization and signaling hubs. The authors use a range of experimental approaches  to show that FER regulates the formation of the FLS2-BAK1 complex, that additional receptors also regulate the stabilization these complexes on membrane as 'nano-domains'. Furthermore, they show that a domain of FER that regulates cell shape is dispensable for the immune response and RALF23 (a ligand for FER) also stabilizes BAK into nanodomains.The story presented shows a logical progression and is based upon the results presented.The major strength of the paper is the methods used. For example, high resolution imaging of the cell surface is utilized to directly visualize the dynamics of these proteins, and this is coupled with single particle tracking analysis, thus providing an accurate picture of these domains and their dynamics. Genetic lines are used to specifically examine the functionality of the proteins and their domains.However, in order to improve the manuscript, I believe the authors should provide further details about the methods used and analysis details. For example, at present many of the figures are presented with statistical analysis to compare experimental conditions, but in the figure legends they state 'similar results were obtained in three independent experiments'. To present the data as robustly as possible, I suggest that the authors should show and perform statistical analysis on these similar experiments. Thus, providing the readers with a chance to evaluate how robust the effect is and to understand how variable the experiments were.A weak experimental point is the examination of protein accumulation on the plasma membrane – which is used to show that the nanodomain results in the stabilization of FLS on the membrane, preventing them from being endocytosed. Here single channel images are used to determine the amount of protein based on fluorescent intensity without further controls . Additionally, it is not clarified if the fer mutants used have altered endocytosis in general.The legends/methods need further details. For example, in figure 1 supplemental 1, it is not clear what the data points are; are they individual tracks, or cells from the same plant? Thus, it is important to clarify further how the analyses was conducted .I am not sure 'propensity' is the appropriate word, perhaps 'property', line 23.In order to appeal to non-specialists, and to aid the readers comprehension, the authors should consider introducing less abbreviations and focusing on only the critical ones. For example, just in the 1st introduction paragraph (lines 41-55) there are 9, thus at present, it is quite hard to follow the text.The authors should change the word static to describe the FLS foci on the cell surface (line 108). The foci are not static as they appear and disappear over time, thus they should consider using the terminology, 'laterally stable foci' or something similar.As much of the analysis of live imaging relies on trackMate, and while the authors detail the settings used, there is no information about how the threshold values was selected. This is important as for example, during the videos, there is bleaching during acquisition which could result in the shortening of tracks. Furthermore, while I understand that is visually easy to show these results with kymographs, the authors should include a histogram of the foci spot lifetimes  to more robustly depict the data.In general, while the videos with tracking are a great addition to the manuscript, at present the fact that the tracks remain after the spots have disappeared is distracting and makes it hard to see the dynamics of the foci. It should be simple enough to change the videos with trackMate , which would greatly improve the usefulness of the tracking videos.The western blots should be quantified to show the results are robust and reproducible. And there are some signals which appear to be saturated.While I understand the focus is on the FLS BAK dynamics, I think it would be interesting to show how specific this interaction is for mediating the formation of the nanodomains. For example, by examining another receptor or cargo in the mutant lines it would tell us if FER is a general nanodomain scaffold protein.Line 114 – authors should state how many frames were combined to create the average projections.There are no scale bars on the kymographs, so it's impossible to know the duration of imaging/tracks/nanodomains.Line 123 – I think the authors mean formation/composition and not localization.Line 145 – reference to figure needs updating.Line 188 – should be mobile rather than labile.For the figures showing a single track as a model, it would be good in include a scale bar to allow the reader to understand the scale of these diffusions/domains.Line 200 – 'deleted' should be mutated, truncated or altered.206 – should be '…can directly monitor the cell wall.'219 – co-jointly should be rewritten to say, '..and together they relay..'.Figure 3 – it would be good to quantify these effects to show how reproducible they are. Maybe for cotyledon – a line profile across the image to show the cell is more wavey? And a density for root hairs over a certain length?If possible, it would be a great addition to the paper to show that dual dynamics of FLS and BAK in the different experimental conditions.Line 869 – reference to figure needs updating.Reviewer #3:eLife-74162) brings an original set of data describing the role of FER receptor kinase in the control of PM organization and dynamics of plant immune receptor kinases FLS2 and its co-receptor BAK1. Using an advanced fluorescence microscopy approach combined with biochemistry and molecular biology, authors show how the perception of plant peptide hormone RALF23 triggers a specific changes in the distribution of receptor kinases FLS2 and BAK1 in cells at the surface of cotyledons (epidermis of 5-day-old young seedlings). Moreover, these changes are shown to be regulated also by other other receptors of FER, i.e. LRX3, LRX4 and LRX5. The formation of the immune receptor kinase complexes with downstream signalling are therefore suggested to be under the control of RALF23 peptide hormone through the action of both FER and LRXs. Moreover, the data allowed authors to conclude that the effects on root hair growth and immune response are uncoupled in the case of both FER and LRXs receptor pathways and that the kinase activity of FER is needed for the inhibitory effect of RALF23 on the immune response, while the role of FER kinase in the pattern recognition receptors-triggered immunity is kinase-independent. The manuscript is very detailed, bringing the description of the nanoscale localization of receptor kinases; however, the biology behind the observed effects is still a point for future research. Results are basically supported by data, although I have some comments on their presentation.The manuscript of Gronnier et al.  that "similar results were obtained in three independent experiments". I think that in the case of quantifications of microscopy images, it would be perfect to understand how observed differences in the dynamics of receptor kinases are robust when analyzed in these three mentioned biological repetitions. It would also be informative to include some rationale on the selection of cells for the analysis, e.g. was the size the criterion or something else?For a broader community of readers, it might be perhaps better to introduce a bit what is that „peptide hormone". I know that authors are very deeply involved in the RALF23 and often simply call this molecule „peptide". However, in the title of this manuscript, the term “peptide hormone" is used, but, the word "hormone" is not used in the manuscript at all. For broader community, this is a bit difficult to follow.I think that for sure the biological implication of this work would be enhanced if data from biological repetitions mentioned in the text would be involved.Kymographic analyses are not described in methods nor in captions. Axes of kymograms shown in the manuscript are not annotated; therefore it is not clear how actually dynamic the processes are. Time scale would help here.In the Figure 1, suppl. Figure 1 the caption is not mentioning the statistics used in this analysis.Line 877 – subscript should be used for numbers in chemical formulas.VA-TIRFM is mentioned by authors as the main microscopy method used in this contribution. I hope I got it right, therefore, the abbreviation TIRFM in all main and supplementary captions should be changed to VA-TIRFM, as well as in the description of the microscopy itself (lines 807 and 862).Line 869 – the reference to the suppl. image is not correct, it should not be Sup Figure 15, but Figure Suppl 6.The quality of language is very good, however, there are some subtle grammar issues, e.g. on line 136 – „BAK1 might dynamically associates with", I found also some typos etc . I did not have time to find all of them, I encourage authors to check it again.In vivo advanced fluorescence GSD microscopy is used here and I appreciate a lot this technique and how it is implemented. It would be perhaps good to discuss how far individual markers characterize the mobility of the structure where it is located  and how far this technique might be taken as the characterization of the mobility of the particular molecule within the particular structure. Essential revisions:From the written reviews and discussion, there was much enthusiasm for the work, but two general concerns emerged: (1) replicated data were not fully analyzed and reported and (2) the ability of a broad audience to engage with and appreciate the detailed work would be enhanced by modifications to the text. From the individual reviews appended below you will find specific examples of places where modifying text or presentation would have the largest impact.1) That "similar results were found among three replicates" is mentioned in nearly every figure, but these replicates are not shown. After a healthy discussion among the reviewers about what was essential to show, all three reviewers concurred that in the cases of single particle tracking and the quantitative imaging of mobility , the data from the three replicates should be analyzed and the differences between experiments reported. This is in part because this paper has the potential to serve as a standard bearer for this technique.We now present data of individual replicate experiment as well as their statistical analysis.In the case of Western blots, showing a single representative blot was fine, but the band intensities should be quantified using standard densitometry scans. We also encourage the authors to include the replicate, uncropped, blots in a data supplement. For the confocal images of leaves in Figure 3C-D, the single set of images is also fine, but additional details about what constitutes a replicate (see comment from Rev 1) is needed. For other phenotypic measurements like seedling fresh weight, reporting the single replicate is fine.We have quantified the bands intensities  of the western blots. We present replicate experiments, and corresponding uncropped blots in supplementary data. We also clarified what constitutes a replicate for Figure 3C and D.2) A weak experimental point is the examination of protein accumulation on the plasma membrane – which is used to show that the nanodomain results in the stabilization of FLS on the membrane, preventing them from being endocytosed. Here single channel images are used to determine the amount of protein based on fluorescent intensity without further controls . Additionally, it is not shown whether fer mutants have altered endocytosis in general. Please either include supporting experiments or modify the text to indicate the limitations of the current study.e.g. Göhre et al., Curr. Biol. 2008; Smith et al., PLoS Pathog. 2014; Wang et al., New Phytologist 2020), and was shown to reveal defect in protein accumulation that could otherwise only be detected by tedious biochemically purification of the plasma membrane . Furthermore, VA-TIRM experiments consistently showed a decrease in FLS2-GFP accumulation at the plasma membrane . Similar to previous studies, we observed that compromised in nanodomain organization correlates with a defect in plasma membrane accumulation, but we do not claim that these observations demonstrate causality. As suggested by Reviewer 2, we now also discuss the potential involvement of the proposed regulation of endocytosis by FER .We think that the use of single channel images to analyze the amount of tagged proteins is well-suited to quantify protein accumulation at the plasma membrane. Indeed, it has previously been used to robustly quantify FLS2 plasma membrane accumulation (3) The manuscript is very detailed as it described the nanoscale localization of receptor kinases, and the jargon and extensive use of abbreviations makes it challenging for readers outside the plant immune signaling world. Additional discussion and presentation of these findings in an integrative model would help to make the details illustrative of general signaling rules and would broaden the impact of this work. In addition, the phrasing of the abstract that includes mention of a "plasma membrane nanoscale landscape" may lead one to expect that paper will focus on the "landscape" of the PM and the structural changes in the PM provoked by the activity of individual receptor kinases. However, the main focus in on the complex dynamics of individual receptor protein kinases with the PM – no less interesting, but not completely aligned with the abstract.We reduced the number of abbreviations. We now further discuss our findings and present a model. We agree that the word ‘landscape’ would imply a broader investigation of the properties of the plasma membrane, and therefore replaced landscape by organization in the abstract and in the title.4) Many of the experimental details or choices are not fully explained; for example, FER effects on cell wall integrity are mostly known from root hair work, but the experiments here focus on cotyledon epidermal cells. Please add some rationale for experimental design choices, be clear about what a "sample" or "replicate" entails, and mention potential limitations of the current work.We now further explain experimental details, and clarified the meaning of sample and replicate in the figure legends and in the method section.FERONIA function in cell wall integrity is well described in cotyledon epidermal cells and was linked to cell morphogenesis  and mechano-sensing . We recapitulated observations related to cell morphogenesis in our laboratory conditions  to provide a clear comparative analysis with the immune signaling outputs.Reviewer #1:How does a protein identified as having a role in many different and seemingly independent plant responses act in specific ways in those responses? An example in plants is the receptor kinase FERONIA (FER) that has been implicated in numerous processes including cell wall integrity signaling and response to biotic and abiotic stresses. Using a series of cell biological and biochemical approaches, the authors show, using the intensively studied FLS2-BAK1 immune receptor complex and its previous identified ligands and signaling potentiators or inhibitors, that different domains and activities of FER have discrete roles in different cellular events. They further show that FER and LRX proteins may affect the mobility of plasma membrane proteins in their nanodomains, though precisely how loss of FER or LRX leads to changes in nanodomain properties is not entirely clear. This detailed work adds interesting and important information about how individual proteins and cellular milieus can alter signaling. Additional discussion and presentation of these findings is an integrative model would help to make the details illustrative of general signaling rules and would broaden the impact of this work outside of the plant immunity niche.From the set-up of this paper, I imagined that the authors would show a unified way in which FER and/or LRXs affected a cellular feature (e.g. nanodomain formation) and this would account for multiple activities. The results of their experiments, however, showed that they can separate activities of FER . I am left not quite knowing how direct FER activity is on FLS2-BAK1 behaviors. I was expecting it to be indirect through some nanodomain organization, but I failed to find this thread followed through. The direct effects of FER are also interesting results, but as an outsider to the PTI and cell wall integrity fields, I find it challenging to synthesize the results into a general overall model. I don't think the authors need more experiments, but I do think a figure that summarizes their updated concept of how FER works would make this paper accessible and appreciated by a wider audience.We thank the reviewer for her/his positive comments. We now further discuss our findings and present a model illustrating main findings and potential future directions.Reviewer #2:[…] In order to improve the manuscript, I believe the authors should provide further details about the methods used and analysis details. For example, at present many of the figures are presented with statistical analysis to compare experimental conditions, but in the figure legends they state 'similar results were obtained in three independent experiments'. To present the data as robustly as possible, I suggest that the authors should show and perform statistical analysis on these similar experiments. Thus, providing the readers with a chance to evaluate how robust the effect is and to understand how variable the experiments were.The manuscript now includes the data and statistical analysis of the independent experiments.A weak experimental point is the examination of protein accumulation on the plasma membrane – which is used to show that the nanodomain results in the stabilization of FLS on the membrane, preventing them from being endocytosed. Here single channel images are used to determine the amount of protein based on fluorescent intensity without further controls . Additionally, it is not clarified if the fer mutants used have altered endocytosis in general.Please see our answer to editorial comment #2.The legends/methods need further details. For example, in figure 1 supplemental 1, it is not clear what the data points are; are they individual tracks, or cells from the same plant? Thus, it is important to clarify further how the analyses was conducted .We now further detail the legends regarding sample size and to what correspond the data points for each graph.I am not sure 'propensity' is the appropriate word, perhaps 'property', line 23.We think that propensity better reflect the dynamic aspect of spatial partitioning.In order to appeal to non-specialists, and to aid the readers comprehension, the authors should consider introducing less abbreviations and focusing on only the critical ones. For example, just in the 1st introduction paragraph (lines 41-55) there are 9, thus at present, it is quite hard to follow the text.We have reduced the number of abbreviations and present a table summarizing them.The authors should change the word static to describe the FLS foci on the cell surface (line 108). The foci are not static as they appear and disappear over time, thus they should consider using the terminology, 'laterally stable foci' or something similar.FLS2 foci are now described as laterally stable foci.As much of the analysis of live imaging relies on trackMate, and while the authors detail the settings used, there is no information about how the threshold values was selected. This is important as for example, during the videos, there is bleaching during acquisition which could result in the shortening of tracks. Furthermore, while I understand that is visually easy to show these results with kymographs, the authors should include a histogram of the foci spot lifetimes  to more robustly depict the data.The thresholds were empirically determined for optimal single particle detection and linking. We do not consider foci spot lifetimes as a relevant metric to describe the lateral organization/dynamics of particles, which is the focus of our study.In general, while the videos with tracking are a great addition to the manuscript, at present the fact that the tracks remain after the spots have disappeared is distracting and makes it hard to see the dynamics of the foci. It should be simple enough to change the videos with trackMate , which would greatly improve the usefulness of the tracking videos.To the contrary, we think that full tracks better reflect the behavior of the single particles.The western blots should be quantified to show the results are robust and reproducible. And there are some signals which appear to be saturated.We now provide quantification of the western blots.While I understand the focus is on the FLS BAK dynamics, I think it would be interesting to show how specific this interaction is for mediating the formation of the nanodomains. For example, by examining another receptor or cargo in the mutant lines it would tell us if FER is a general nanodomain scaffold protein.We share the interest of the reviewer, and are actively working to determine whether the FER function described in the present manuscript applies to for additional RK-mediated signaling pathways. However, such detailed investigation is far reaching and beyond the scope of our current manuscript.Line 114 – authors should state how many frames were combined to create the average projections.This information is now indicated in each figure legend.There are no scale bars on the kymographs, so it's impossible to know the duration of imaging/tracks/nanodomains.We now provide scale bars for the kymographs.Line 123 – I think the authors mean formation/composition and not localization.We changed localization to organization. Thank you.Line 145 – reference to figure needs updating.This is now corrected. Thank you.Line 188 – should be mobile rather than labile.We assumed that the reviewer refers to L118, and changed labile to mobile.For the figures showing a single track as a model, it would be good in include a scale bar to allow the reader to understand the scale of these diffusions/domains.These models correspond to graphical illustration summarizing our observations but not to experimental single particle trajectories. We prefer not to include a scale bar, as it may be confusing.Line 200 – 'deleted' should be mutated, truncated or altered.This is now corrected. Thank you.206 – should be '…can directly monitor the cell wall.'We are here unsure which sentence the reviewer refers to.219 – co-jointly should be rewritten to say, '..and together they relay..'.We are here unsure which sentence the reviewer refers to.Figure 3 – it would be good to quantify these effects to show how reproducible they are. Maybe for cotyledon – a line profile across the image to show the cell is more wavey? And a density for root hairs over a certain length?We think that these observations are already very clear and are certainly reproductible.If possible, it would be a great addition to the paper to show that dual dynamics of FLS and BAK in the different experimental conditions.This remains technically challenging and would require further extensive work.Line 869 – reference to figure needs updating.This is corrected, thank you.Reviewer #3:[…] I feel that this report needs more attention to the biology itself. For the broader community, it would be perfect to understand in what process the mechanism described here is crucial. Therefore, I feel that authors would much improve this manuscript if they would be able to defend why they use epidermal lobed cells in 5-day-old seedlings. I know that there might be plenty of technical reasons, previous work, etc., but biologists would ask about it; considering that effects on cell growth are shown in root hairs, while all immune responses are studied in cotyledon epidermal lobed cells. The introduction on why it is actually so important to study described processes in cotyledons would help.Please see our answer to the editorial comment #4Perhaps I am wrong, but the "plasma membrane nanoscale landscape", as mentioned in the last sentence of the abstract, is related to the nanoscale organization of receptor kinases studied here, not the "landscape" of the PM itself. Of course, PM is extremely dynamic, but this manuscript is not focused on the understanding of PM structural changes provoked by the activity of individual receptor kinases. It is rather focused on surprisingly complex dynamics of individual receptor protein kinases with the PM. This I feel needs to be presented in a clearer form.We agree and have replaced landscape by organization in the abstract.Statistics is provided for the majority of analyses. However, authors mention in numerous cases  that "similar results were obtained in three independent experiments". I think that in the case of quantifications of microscopy images, it would be perfect to understand how observed differences in the dynamics of receptor kinases are robust when analyzed in these three mentioned biological repetitions. It would also be informative to include some rationale on the selection of cells for the analysis, e.g. was the size the criterion or something else?We thank the reviewer for her/his proposition and now provide data from individual experiments and their statistical analysis. Concerning the observation by VA-TIRFM: after placing the center of cotyledons of 5-day-old seedlings (entire seedlings were mounted between cover slips and slides) at the center of the objective lens, we screened, by moving in x and y, using pre-defined and fixed laser angle and power, for cells surfaces in distance range of the evanescent wave without additional a priori selection criterions.For a broader community of readers, it might be perhaps better to introduce a bit what is that „peptide hormone". I know that authors are very deeply involved in the RALF23 and often simply call this molecule „peptide". However, in the title of this manuscript, the term “peptide hormone" is used, but, the word "hormone" is not used in the manuscript at all. For broader community, this is a bit difficult to follow.I think that for sure the biological implication of this work would be enhanced if data from biological repetitions mentioned in the text would be involved.We now introduce RALF23 as a peptide hormone in the introduction. Thank you.Kymographic analyses are not described in methods nor in captions. Axes of kymograms shown in the manuscript are not annotated; therefore it is not clear how actually dynamic the processes are. Time scale would help here.We have added scale bars for all kymographs. The kymographs were generated using “orthogonal views” . This information has been added to the material and methods section. Thank you.In the Figure 1, suppl. Figure 1 the caption is not mentioning the statistics used in this analysis.This is now implemented. Thank you.Line 877 – subscript should be used for numbers in chemical formulas.This is now corrected. Thank you.VA-TIRFM is mentioned by authors as the main microscopy method used in this contribution. I hope I got it right, therefore, the abbreviation TIRFM in all main and supplementary captions should be changed to VA-TIRFM, as well as in the description of the microscopy itself (lines 807 and 862).This is now corrected; we replaced TIRFM by VA-TIRFM throughout the text.Line 869 – the reference to the suppl. image is not correct, it should not be Sup Figure 15, but Figure Suppl 6.This is now corrected. Thank you.The quality of language is very good, however, there are some subtle grammar issues, e.g. on line 136 – „BAK1 might dynamically associates with", I found also some typos etc . I did not have time to find all of them, I encourage authors to check it again.We have now checked and corrected the manuscript for grammar issues. Thank you.In vivo advanced fluorescence GSD microscopy is used here and I appreciate a lot this technique and how it is implemented. It would be perhaps good to discuss how far individual markers characterize the mobility of the structure where it is located  and how far this technique might be taken as the characterization of the mobility of the particular molecule within the particular structure.Thank you for your interest. We haven’t performed such a comparative analysis and are therefore unfortunately not in the position of evaluating it limits.


Background: Macrovascular complications occur very frequently in patients with type 2 diabetes mellitus (T2DM) with a high mortality rate, due to the development of cardiovascular disease (CVD), such as stroke, atherosclerosis acceleration, and atrial fibrillation. T2DM is a significant risk factor for CVD and has become the leading cause of death. The purpose of this study was to detect the early risk of macrovascular complications by using the ankle brachial index (ABI) as a marker.Methods: This study was an analytic study with a cross-sectional approach. The study population was patients with T2DM from several primary health care centers in Medan. In total, 89 subjects who met the inclusion and exclusion criteria were recruited with consecutive sampling. ABI was determined as the ratio of systolic blood pressure in the brachial artery to the posterior tibial artery after the subjects had been relaxed and felt comfortable in a supine position. Examination of vitamin D and lipid profile was derived from examination of venous blood.  Data were processed using SPSS and analyzed with one-way ANOVA.Results: The study found that there was a relationship between LDL-C, triglyceride, and vitamin D (25OH-D) based on the ABI (p > 0.05).Conclusions: ABI can be used for an early detection of macrovascular complications. Apart from being easy to perform, ABI was non-invasive. Some other risk factors that can also be used to assess complications and have relationships with ABI were LDL-C, triglyceride, and vitamin D (25OH-D). Complications in T2DM patients can be prevented with reasonable blood sugar control and lifestyle changes. Education and motivation need to be given to patients so that they become more independent in controlling their disease and improving their quality of life. T2DM is a significant risk factor for CVD and has become the leading cause of death, whereby almost 50% of the total deaths are due to this disease.1 It is estimated that the mortality rate of CVD increases almost threefold in T2DM.2 Besides CVD complication, peripheral arterial disease (PAD) has also become a serious condition of macrovascular complications. PAD is characterized by narrowing peripheral arteries due to atherosclerosis, and it generally occurs in the leg arteries. Many patients with T2DM are suffering from PAD, and as this disease is non-symptomatic, it is undetected. PAD is one of the T2DM complications associated with CVD. Atherosclerosis is the most common and important cause of PAD. Dyslipidemia, smoking, hypertension, and T2DM are also known as risk factors for PAD, CVD and coronary artery disease.7 Ankle brachial index (ABI) is a non-invasive tool for detecting vascular status and for measuring lower leg arteries versus upper leg arterial health status.8 ABI is the ratio between the lower limb (ankle) systolic blood pressure and the upper limb (upper arm) systolic blood pressure. ABI reflects vascular resistance, which explains the main factors: the blood vessel diameter, which can be narrowed either from internal factors  or from external factors, such as compression by soft tissue.9 ABI examination is a non-invasive gold standard measurement to detect PAD; consequently, the examination is highly recommended for patients with T2DM and others at risk of suffering from PAD. An early detection can reduce the risk of more severe complications, such as neuropathy and CVD disorders.5 The purpose of this study was to detect the early risk of macrovascular complications by using the ABI as a marker.Patients with type 2 diabetes mellitus (T2DM) are at risks of short- and long-term complications. Long-term complications include macrovascular and microvascular diseases.This was an analytical study with a cross-sectional approach. The study had the ethical approval from the Research Ethics Commission of Universitas Sumatera Utara .10 A total of 89 subjects that met the inclusion and exclusion criteria were recruited with consecutive sampling technique. Patients who participated in this study were recruited from three primary health services in Medan . For measuring the blood pressure of the brachial artery, the cuff was placed at the upper arm. The systolic pressure was considered as the first occurrence of rhythmic sounds heard with stethoscope placed on the antecubital fossa. For the posterior tibial blood pressure, the cuff was placed at the ankle, the systolic pressure was considered as the first pulse was palpable on the dorsum pedis during the cuff deflation. ABI lower than 0.90 was considered abnormal, which was then classified into 0.41–0.90 as a mild to moderate decrease in blood flow, while <0.40 as a severe decrease in blood flow.12Most of the data collection was done between 1813 and triglyceride (TG) level was measured using the GPO–Trinded method.14Hydroxyvitamin D (25OH-D) level and lipid profile  were analyzed from the venous blood samples (5 ml) drawn after 10 hours fasting. Hydroxyvitamin D (25OH-D) were measured through enzyme-linked immunosorbent assay (ELISA) with human vitamin D ELISA kit . Serum for lipid profile was processed by and Auto Analyzer (Indiko Thermo Scientific™) and the total cholesterol (TC) level was measured using cholesterol oxidase method, HDL-C and LDL-C levels analyzed by enzymatic colorimetric method,15Determination of the patient's nutritional status was done by using the Body Mass Index (BMI), which is defined as body weight in kilograms divided by the square of body height in meters (kg/m2), which is then adjusted to the World Health Organization classification.The data was analyzed with SPSS 22 for Windows and then shown in tables. The data under consideration was complete data; any incomplete data was deleted. We included all participants who satisfied the inclusion criteria until the minimal number of samples was reached.The Kolmogorov Smirnov test (p > 0.05) was used to estimate the average of the normal distribution of the sample data. The normality test findings were utilized in the following analysis: parametric analysis was done if the distribution was normal; otherwise, non-parametric analysis was used. To show how ABI influenced average age, diabetes duration, blood pressure, total cholesterol, HDL-C, LDL-C, TG, and vitamin D (25OH-D) (p < 0.05), a one-way ANOVA was used. The method was then followed by least significant difference or Bonferroni testing if a significant result was obtained (p < 0.05).Most patients were female , and from the age group of 46–55 years . Normal nutritional status was noted in 44 subjects (49.4%). Based on the duration of illness, more than half of the subjects  had T2DM for 1–5 years .16 A low ABI value reflects atherosclerosis in the vessel wall, resulting in a decrease of perfusion and arterial circulation to the distal extremities.17 This reduction in perfusion is usually characterized by loss of peripheral pulses, intermittent claudication, and complicated infection of the legs.18 The pathogenesis of atherosclerosis in PAD is the same as that of the coronary arteries. The lesion in the segment creates stenosis or occlusion that usually takes place in the large or medium-sized vessels. The development of these lesions become atherosclerotic plaques with calcium build-up, thin tunica media, destruction of muscle and elastic fibers, and fragmentation of the elastic internal lamina; and thrombus consisting of platelets and fibrin may happen.19Diabetes with vascular inflammation has become a risk factor for atherothrombotic diseases, such as PAD.20 According to a study by Hiramoto (2014), after 30 months of observation, the ABI value decreased in patients with T2DM by 20.7%, with 5% of the patients showing PAD symptoms.19 The factors causing the decrease in ABI were influenced by age, gender, high HbA1c, high serum creatinine, high LDL-C, and retinopathy.21Hypertension and diabetes increase the risk of the development of macrovascular and microvascular complications.Dyslipidemia is a metabolic disorder associated with diabetes. It is characterized by a spectrum of quantitative and qualitative changes in lipids and lipoproteins, including hypertriglyceridemia, decreased concentrations of high-cholesterol lipoprotein (HDL), and elevated LDL.22 Various studies have proven that the risk factors for cardiovascular disease and death from coronary heart disease are directly related to the concentration of cholesterol in the blood.23 Hypercholesterolemia can directly cause endothelial dysfunction through the production of reactive oxygen species, which converts low-density lipoprotein (LDL) into oxidized low-density lipoprotein (ox-LDL).24 LDL-C and TG are markers that have been used to assess the occurrence of atherosclerosis (atherogenic dyslipidemia).26 Atherogenic index of plasma, on the other hand, is an instrument that has been used to assess the occurrence of atherosclerosis by using TG level as a marker.28 Ultimately, small dense LDL-C leads to an increased risk for the development of CVD.7 Complications such as uncontrolled blood sugar levels, i.e., random blood sugar level ≥ 200 mg/dL, and fasting blood sugar level ≥ 126 mg/dL occur in T2DM patients within an average period of 5–10 years.32 The low ABI is influenced by irregular consumption of anti-hyperglycemic drugs, irregular physical activity, irregular foot care, and irregularity in the T2DM diet.33This study indicated that there was no relationship between the length of illness and age with ABI. The results were not in line with other studies that showed that age and length of illness affecting ABI, and that this risk increased in patients aged ≥50 years and a history of diabetes-associated atherosclerosis. and there is a strong association between vitamin D deficiency and the prevalence and severity of PAD,37 however, other studies have demonstrated that serum levels of Vitamin D are not associated with arterial stiffness or PAD.36 Previous studies have reported that vitamin D has a preventive role in patients with CVD.39This study shows that there was relationship between vitamin D and ABI. Vitamin D is associated with arterial atherosclerosis, which underlies an increased risk of CVD,35 T2DM and vascular inflammation have become risk markers and even risk factors for atherothrombotic disease, including PAD. T2DM increases the process of atheroma formation. There is an increase in histamine levels in plasma and cells in patients with T2DM and PAD, which can lead to an increased endothelial permeability.40 The next process is the migration of T-lymphocytes into the intima and the increase in the secretion and the activation of cytokinesis. Monocytes/macrophages ingest oxidized LDL molecules and turn them into foam cells, where the accumulation of these cells will form fatty streaks, which are the precursors of atheroma. Atheroma plaque will become unstable because endothelial cells in patients with T2DM secrete cytokines that inhibit collagen production by smooth muscle cells of the blood vessel. Metalloproteinases are also released by these inflammatory cells, which can destroy the collagen fibrous cap plaque atheroma, increasing the tendency for plaque rupture and thrombus formation.16 Systemic inflammation has also been evident to increase insulin resistance. T2DM is an inflammatory condition, and vitamin D may reduce insulin resistance by reducing the risk of inflammation.This vitamin inhibits the renin–angiotensin–aldosterone system and modulates macrophage activity and cytokine production.41 In populations with vitamin D deficiency and impaired glucose tolerance and T2DM, vitamin D supplementation can improve insulin secretion, glucose tolerance, and decrease HbA1c levels. The provision of vitamin D supplementation in healthy adults with this deficiency has shown to improve insulin sensitivity by 60%, which is better than rosiglitazone and other metformin therapy.42There is an association between low vitamin D levels and T2DM, and impaired glucose tolerance.43The abnormalities in the function of endothelial cells and vascular smooth muscle and the tendency to thrombosis are affected by atherosclerosis and its complications. Endothelial cells can regulate blood vessel function and structure because of the strategic anatomical position between the blood vessel walls and blood flow. Under normal circumstances, many active substances are synthesized and released by endothelial cells to maintain blood vessel homeostasis and to regulate blood flow and nutrients to the tissues while preventing thrombosis and leukocyte diapedesis.44Individuals with low serum vitamin D levels have an increased risk of dyslipidemia, insulin resistance, and T2DM. The effect of vitamin D on the improvement of T2DM can be directly obtained through insulin action. Vitamin D enhances insulin response to glucose transport by stimulating insulin receptor expression in peripheral tissues. Moreover, vitamin D improves the level and the integrity of lamellae in the aortic medium tunica and prevents fragmentation of the aortic elastic fibers. Vitamin D supplementation in adult patients with T2DM significantly improves lipid profiles. Therefore, it can be concluded that fat metabolism disorders are caused by vitamin D deficiency.45The limitation of this study was that it did not distinguish patients with a history of smoking from non-smoking individuals. The results of other studies also say that a history of or smoking affects blood vessels because it causes atherosclerosis in blood vessels that contributing significantly to the ABI value, and smoking can significantly increase DM patients experiencing diabetic peripheral neuropathy.Early detection of macrovascular complications is vital to prevent morbidity and mortality in T2DM patients. ABI can be used for the early detection of macrovascular complications. Besides being simple and non-invasive, the ABI is also entirely accurate for detecting macrovascular complications in T2DM patients. The results demonstrated that there was a correlation between the ABI value and the average LDL-C level, triglycerides, and vitamin D.https://doi.org/10.6084/m9.figshare.14915724.v1.46Figshare: Laboratory Result of type 2 DM Patients,•Data file This project contains the following underlying data:Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the This study is simple yet very powerful in advocating the use of Ankle Brachial Index as a risk measurement for peripheral arterial disease as a manifestation of macrovascular problem. The predictive capability of ABI to measure the risk for peripheral arterial disease is also consistent with the level of LDL-cholesterol and triglycerides known to be risk factors for cardiovascular diseases. Of particular interest, which should be emphasized in the discussion is concerning the positive association between the level of vitamin D and the level of peripheral arterial disease. This is a very important finding now when many people consume high dose of vitamin D to prevent severe COVID-19. Evidence-based explanation of the negative effect of higher level of vitamin D need some more elaboration.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:My areas of research is epidemiology as applied to family medicineI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The manuscripts could be improved if the authors include detailed information in methods with sufficient data and supporting evidence in results and discussion sections.The introduction needs to be justified, with limited evidence to show the magnitude of the problem.The conclusion could be more concise and better worded study implications.Language-wise is good.et al. (2017[ref 1]),  Angellottiet al. (20172) and Palomer et al. (20083).Limited references were used in supporting the findings. See SilvaIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Community Nutrition, Functional Foods & NutraceuticalsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Bronchopulmonary dysplasia (BPD) is a severe lung disease that affects preterm infants receiving oxygen therapy. No standardized, clinically-relevant BPD model exists, hampering efforts to understand and treat this disease. This study aimed to evaluate and confirm a candidate model of acute and chronic BPD, based on exposure of neonatal mice to a high oxygen environment during key lung developmental stages affected in preterm infants with BPD. Neonatal C57BL/6 mouse pups were exposed to 75% oxygen from postnatal day (PN)-1 for 5, 8, or 14 days, and their lungs were examined at PN14 and PN40. While all mice showed some degree of lung damage, mice exposed to hyperoxia for 8 or 14 days exhibited the greatest septal wall thickening and airspace enlargement. Furthermore, when assessed at PN40, mice exposed for 8 or 14 days to supplemental oxygen exhibited augmented septal wall thickness and emphysema, with the severity increased with the longer exposure, which translated into a decline in respiratory function at PN80 in the 14-day model. In addition to this, mice exposed to hyperoxia for 8 days showed significant expansion of alveolar epithelial type II cells as well as the greatest fibrosis when assessed at PN40 suggesting a healing response, which was not seen in mice exposed to high oxygen for a longer period. While evidence of lung inflammation was apparent at PN14, chronic inflammation was absent from all three models. Finally, exposure to high oxygen for 14 days also induced concurrent outer retinal degeneration. This study shows that early postnatal exposure to high oxygen generates hallmark acute and chronic pathologies in mice that highlights its use as a translational model of BPD. Bronchopulmonary dysplasia (BPD) is the most common respiratory disorder affecting premature infants provided long-term oxygen therapy and respiratory support . AdvanceNumerous high oxygen schemes have been used to model BPD in rodents. These models have contributed to our understanding of mechanisms responsible for the development of this condition, such as the role of oxidative stress and inflammation , and theIn previous studies, supraphysiological levels of oxygen (85–100%) have been administered over a long period of time , 14. SucThe appearance of the alveolar deformities associated with BPD is largely connected with the interruption to normal lung development . This isThe purpose of this study was to validate whether acute high oxygen exposure received during key periods of lung development would lead to long-term pulmonary changes. Given that the aforementioned multi-model study only assessed lung damage at day 14, in this study, we have focused on evaluating different parameters of lung pathology that may arise in adulthood, and unlike other multi-model studies, the assessment of outer retinal changes as a common co-morbidity of BPD were also prioritized in this study. Herein, we demonstrate that prolonged oxygen exposure in early neonatal life leads to sustained alveolar deterioration in adulthood, as well as defects in retinal tissue, and therefore is a suitable model for early and later life studies of BPD and research into the long-term impact of oxygen toxicity to the eye.C57BL/6 mice were used for this study and were purchased from Alfred Animal Services at the Alfred Research Alliance. Neonatal mice together with their dam, in litter sizes of 6–7 pups , were exposed to 75% oxygen within 12 h of birth (defined as PN1) up to PN5, PN8, or PN14, and were cycled with 21% oxygen (room air) for 3 h per day to prevent oxygen toxicity to the dam . Followihttp://imagej.nih.gov/ij) and analyzed using a published script  or Picrosirius Red (PR). H&E stained sections were imaged using an Olympus BX-51 bright field microscope equipped with a DP-70 color camera and 10x and 40x objectives . Quantitation of alveolar airspace diameter and septal wall thickness was determined using the mean linear intercept method and alveolar septal wall thickness technique, as previously described . Investid script  with minFor immunohistochemistry, deparaffinized sections underwent antigen-retrieval with DAKO Target Retrieval Solution . Sections were blocked for 30 min with 5% BSA and incubated for 3 h at room temperature with 1:500 dilution of anti-mouse CD45  to stain all immune cells. Control sections had primary antibody substituted with PBS. Staining was revealed by incubation with 1:500 HRP-conjugated secondary antibody  for 1 h and color development with diaminobenzidine chromogen solution . Slides were counterstained with hematoxylin. Cells expressing CD45 were labeled brown, while negative cells were stained blue. Four randomly selected microscopic fields (40x) of lung tissue per mouse were used to calculate the percentage of CD45 positive cells/total cells as described . On sepa2O. The flexiware software (v8.0.4) was used to perform respiratory system mechanics, as previously detailed . Following confirmation of deep anesthesia, mice underwent tracheostomy and a 19G cannula was inserted before being connected to an animal ventilator . Mice were mechanically ventilated  and a positive end-expiratory pressure (PEEP) was set at 3 cm Hdetailed . A negatParaffin-embedded eyes were sectioned at 3 μm thickness and every 20th section was stained with H&E. Four photomicrographs (x10) of each cross-section were captured across the full circumference of the eye and the average diameter (in μm) per section for each eye was determined to obtain choroidal thickness as previously described . InvestiT-test (Mann-Whitney) in GraphPad Prism software ; P < 0.05 was considered statistically significant. Unmarked bars on figures indicate that no significance was achieved.Values are presented as median ± IQR. Results from individual models were compared to the room air controls using the non-parametric unpaired To define the best experimental model that most closely recapitulates BPD, we trialed three oxygen exposure protocols . For eacIn mice, the bulk of alveolarization in the lung is complete by PN14 , formingIt is now well-appreciated that preterm infants diagnosed with BPD have an increased susceptibility to other lung diseases in later life, including COPD , 7. To eA phenotype of “old” BPD is the presence of extensive parenchymal fibrosis, however this trait is significantly milder in the lungs of infants with newly diagnosed BPD due to the changes in respiratory care implemented in the NICU over the last few decades , 30. ThuHyperoxia has been associated with impaired AEC development, promoting an enlarged and simplified lung structure that is commonly observed in BPD , 31. To In order to investigate the effect of high oxygen exposure on lung inflammation in the neonate, immunohistochemical staining for CD45-expressing leukocytes was performed. In the normal developing lung at PN14, ~20% of the cells present were CD45+ leukocytes, which were mostly found within the alveolar walls, around small vessels and occasionally also in the airspaces . In all To determine if the high oxygen insult utilized in the three different models led to sustained inflammation in the lung parenchyma, immunostaining was performed on lung sections from 40-day-old mice. At PN40, proportions of CD45+ leukocytes in all high oxygen exposure models were comparable with the room air control mice  indicatip = 0.0513) and 8 days (p = 0.0728) but it was significantly decreased in mice exposed to high oxygen for 14 days compared to room air control mice . To evaluate the impact of neonatal high oxygen exposure on the thickness of the outer retina, H&E-stained eye sections from 40-day-old mice were examined. At PN40, there was a trending decrease in choroidal thickness in mice exposed to high oxygen for 5  for 14 days or longer has been reported to result in the development of extensive fibrosis in the lung parenchyma , 34–36. Targeting oxygen exposure within the critical window of lung development in experimental models is an important consideration to maintain clinical relevance to human BPD. Importantly, the saccular and alveolar stages of lung development appear to be most impacted during oxygen administration in preterm infants in NICUs . The mouAs expected, 14 days of high oxygen exposure generated the greatest degree of alveolar dysmorphogenesis in the neonatal lung, which manifested as severe pulmonary emphysema in the young and mature adult. The impact of this structural deterioration on lung mechanics was evident by the impaired respiratory function in mature adults exposed to this oxygen scheme. Taken together, these findings indicate that prolonged high oxygen exposure during the two final lung developmental stages in mice promotes lung injury that approximates that seen in preterm infants with BPD, and may even capture those infants that go onto to develop COPD in later life. This model will therefore also be useful to unravel mechanisms that underpin chronic lung conditions that have origins in early life. The ability of the 14-day exposure regime to generate long-term lung damage and functional decline within a relatively short time frame compared to current models in use , 43, alsInflammation is also recognized to contribute to the deterioration of the alveolar structure in the neonatal lung following a high oxygen insult . Airway A common clinical co-morbidity of infants diagnosed with BPD is ROP . The retIn the absence of a cure for BPD, there is a need to identify the mechanisms underlying this debilitating condition. Accordingly, the development of a benchmark model of BPD will allow for greater comparison of results amongst research groups and advances in the understanding of the disease. The current study describes a model of oxygen-induced BPD, comprised of a 14-day exposure to 75% oxygen, initiated within 12 h of birth, which was associated with features of contemporary BPD in neonatal mice. Moreover, the model was associated with the impaired development of alveoli in adult mice that resembled COPD encountered by adult survivors of BPD alongside outer retinal decay, which is a distinguishing feature of this model. These findings provide a distinct advantage, suggesting that this model could be a key translational tool for the trialing of new treatments which may advance therapeutic strategies to improve the care and long-term outcome for vulnerable premature infants. We advocate that the standardized PN1-14 model described herein may facilitate studies into the early life impact of high oxygen-exposure but also expedite investigations into the lifelong effect of oxygen insults received during the neonatal period.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Alfred Research Alliance Animal Ethics Committee .MH and LW conceived the study, designed research, and analyzed data. LW performed experiments. PW supplied an integral piece of equipment, provided critical intellectual content, and important manuscript revisions. CJ contributed a new analytic tool. ET provided valuable insight and made important intellectual contributions. LW and MH wrote the paper. All authors provided editorial comments and gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Adenocarcinoma of the esophagus has been on the rise lately. Increase in mortality due to a paucity of efficacious drugs for this cancer prompted us to discover molecular signatures to combat this malady. To this end, we chose resveratrol—a polyphenol and studied its impact on three esophageal adenocarcinoma cell lines  by multilevel profiling. Here, we show the impact of resveratrol on the viability of the three cell systems studied, at the cellular, molecular level and by proteomic analysis. Impact on programmed cell death pathway resulting in an increase in apoptotic and caspase-positive cells were observed. Decrease in Bcl2. levels and impact on reactive oxygen species (ROS) was also observed. Moreover, proteomic profiling highlighted pivotal differentially regulated signaling molecules. Notably, the downregulation of Ku80 by resveratrol could be harnessed for chemo-radiation therapy to prevent DNA break repair after radiation therapy. Additionally, protein profiling has shed light on molecular and immune-modulatory signatures with implications for discovering novel treatments such as chemo-immunotherapy.Deregulation of signaling pathways due to mutations sets the cell on a path to neoplasia. Therefore, recent reports of increased mutations observed in esophageal tissue reflects the enhanced risk of tumor formation. In fact, adenocarcinoma of the esophagus has been on the rise lately. Increase in mortality due to a paucity of efficacious drugs for this cancer prompted us to discover molecular signatures to combat this malady. To this end, we chose resveratrol—a polyphenol with anticancer property—and studied its impact on three esophageal adenocarcinoma cell lines  by multilevel profiling. Here, we show the impact of resveratrol on the viability of the three adenocarcinoma esophageal cell systems studied, at the cellular level. Furthermore, an analysis at the molecular level revealed that the action was through the programmed cell death pathway, resulting in an increase in apoptotic and caspase-positive cells. The impact on reactive oxygen species (ROS) and a decrease in Bcl2 levels were also observed. Moreover, proteomic profiling highlighted pivotal differentially regulated signaling molecules. The phenotypic effect observed in resveratrol-treated esophageal cells could be due to the stoichiometry per se of the fold changes observed in entities of key signaling pathways. Notably, the downregulation of Ku80 and other pivotal entities by resveratrol could be harnessed for chemo-radiation therapy to prevent DNA break repair after radiation therapy. Additionally, multilevel profiling has shed light on molecular and immune-modulatory signatures with implications for discovering novel treatments, including chemo-immunotherapy, for esophageal adenocarcinomas which are known to be aggressive cancers. Organs such as the skin and cells of the hematopoietic system are known to be prone to mutations; however, a key report on the assessment of mutations points to the esophagus as an additional organ vulnerable to somatic as well as cancer-related mutations . UltimatRecently, natural products with anticancer activity have been sought after to treat various types of diseases, including cancers, because of their minimal side effects. Certain plants such as grapevines are known to synthesize non-flavonoid phenolic compounds, these molecules are categorized as phytoalexins and one such compound is resveratrol . In cervical cancers, this molecule is shown to induce apoptosis as well as downregulate the antiapoptotic molecule Bcl2 . FurtherResveratrol’s action is multifaceted including modulation of key transcription factors . FurtherResveratrol was purchased from Sigma-Aldrich. Stock solution of Resveratrol (100 mM in DMSO) was used for the study. Assay kits for Muse flow cytometry were supplied by EMD Millipore .2.Human esophageal adenocarcinoma cell lines, i.e., OE33, OE19 and FLO-1 cell lines, were purchased from Sigma-Aldrich. OE33 and OE-19 cells were grown in RPMI medium supplemented with 10% fetal bovine serum, 1% Penicillin/Streptomycin and 2 mM glutamine. FLO-1 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% Penicillin/Streptomycin and 2 mM glutamine. Cultures were incubated at 37 °C in an atmosphere of 5% CO50 was determined and the concentrations of resveratrol were plotted versus the values obtained from the CCK8 assay. Three independent experiments were performed for each of the cell lines.Impact of resveratrol on cell viability was assessed by employing the published protocols ,19. Esse50 concentration of each cell line) with untreated controls maintained in parallel. After 72 h of incubation at 37 °C in an atmosphere of 5% CO2, adherent cells were collected by trypsinization and further analysis was conducted. Cells were pooled together from triplicate treatments and were left on ice until analysis.Flow cytometry experiments were performed as per previously published procedures with some modifications ,19. Assa2. This was followed by Muse Caspase 7-AAD working solution and incubation at room temperature for 5 min, protected from light. Analysis for each assay was done as per the manufacturer’s instructions.The following flow cytometric assays were conducted using a Muse Cell Analyzer: (1) Annexin V assay (MCH100105), (2) Bcl2 Dual detection activation assay (MCH200105), (3) Multicaspase assay (MCH100109) and (4) Oxidative stress assay (MCH100111). Assays were performed as per manufacturer’s protocols (Millipore-Sigma). Essentially, for the Annexin V assay, cells were stained with Annexin V & Dead Cell Reagent for 20 min at room temperature and protected from light. For the Bcl2 activation assay, cells were fixed using the Muse fixation buffer and permeabilized using ice-cold Muse permeabilization buffer. The fixed cells were stained with anti-Bcl2, PECy5 antibody for 30 min at room temperature in the dark. To determine whether caspases are involved, Muse Multicaspase Reagent working solution was added to cells and then incubated for 30 min at 37 °C in an atmosphere of 5% CO50 concentrations. Control cultures (no treatment) were maintained in parallel. Essentially, for protein profiling, the following procedures were employed.Proteomic analysis was performed by 2D DIGE and Mass spectrometry, which was performed by Applied Biomics Inc.  employing previously published methodology ,20,21. Og for 30 min at 4 °C. The supernatant of the sample was collected and then diluted in 2D-lysis buffer to achieve a protein concentration of 6 mg/mL.Cells from control samples and resveratrol-treated samples were collected, washed with 1X PBS and then stored at −80 °C prior to sending the samples to Applied Biomics,  . Cells were lysed in 2D cell lysis buffer consisting of 30 mM Tris-HCl-pH 8.8, 7 M urea, 2 M thiourea and 4% CHAPS, and then sonicated on ice and left on a shaker for 30 min at room temperature. Next, the cell suspension was centrifuged at 25,000× The CyDye labeling protocol employed was essentially as described previously ,20,21. Pp-value was set to 0.05.One-way ANOVA analysis with Dunnett’s multiple comparison test analysis, with alpha value set to 0.05 was performed. For the Muse assays, an ANOVA was done with Dunnett’s multiple comparison for both Annexin and MultiCaspase assays. ROS and Bcl2 data analysis was performed by ANOVA. For all the assays, the 50 was calculated to be 100 μM for OE33, 50 μM for OE19 and 40 μM for FLO-1.To assess the anticancer potential of resveratrol on the human esophageal adenocarcinomas; OE33, OE19 and FLO-1 cells were treated with various concentrations of resveratrol and incubated for 72 h. Resveratrol exhibited dose-dependent inhibition of cell viability in all the three cell systems, as shown in 50 concentration of resveratrol (100 μM) for 72 h and compared with the control group. Representative scatter plots of three independent trials of the control group and the treated group are shown in 50 concentration of 50 and 40 μM, respectively, and analyzed by Annexin V Muse flow cytometric analysis. For OE19 cells, representative scatter plots of four independent trials of the control group and the treated group are shown in On observing cell death caused by resveratrol, we proceeded to understand its mechanism of action. Programmed cell death pathway is one of the major pathways induced by anticancer agents. To assess if programmed cell death pathway was induced by resveratrol in each of the cell system, we opted to perform flow cytometric analysis, because this assay is reported to be a sensitive assay. Annexin V Muse flow cytometric assay was performed by treating OE33 cells with the IC50 concentration observed for each of the cell lines. Caspase activation was monitored by Muse flow cytometry and Muse MultiCaspase assay. Three independent trials were performed and representative scatter plots of the control group and the resveratrol-treated group (100 μM) for OE 33 cells are shown in 50 concentration of 50 and 40 μM, respectively, and analyzed by Muse flow cytometry and Muse MultiCaspase assay. Representative scatter plots of four independent trials of the control group and the treated group are shown in We sought to examine if caspases, the key players in the apoptotic pathway, were involved in causing cell death. As it has been reported that a number of caspases play a role in causing apoptosis, we decided to assay for the involvement of multiple caspases. To this end, OE33, OE19 and FLO-1 cells were treated with resveratrol at the IC50 concentration of resveratrol and production of reactive oxygen species was assessed by using the Oxidative Stress kit and Muse Cell Analyzer. The representative profile of three independent trials of ROS assessment in OE33 control cells is shown To ascertain if reactive oxygen species (ROS) was contributing to apoptosis occurring on resveratrol treatment, we monitored for the increase in the generation of ROS by utilizing the Muse oxidative stress assay. Cells were treated with ICBcl2 is a major player in the cell death/cell survival pathway, and is known to be a key molecule in various cancers; thus, we investigated if Bcl2 was affected when treating OE33, OE19 and FLO-1 cells with resveratrol. Bcl2 levels were monitored by a Muse cell analyzer using the Bcl2 assay kit. Representative scatter plot of the OE33 control sample is shown in 50 concentration of 100 μM, 50 μM and 40 μM, respectively, and were processed in an identical manner. Moreover, based on the fold changes obtained, the upregulated and downregulated proteins were selected for further proteomic analysis by mass spectrometry.To gain knowledge of more entities involved in the multifaceted action of resveratrol, we opted to perform a 2-DIGE and mass spectrometric analysis, as this method of analysis could shed light on more molecules involved in the action of resveratrol. The rationale for employing 2-DIGE and mass spectrometric analysis was that this method of protein profiling is highly sensitive as specific peptides of proteins are assessed by mass spectrometry and the accuracy of detection is in the femtomol range. Two-dimensional gel electrophoresis and mass spectrometric analysis were performed with samples obtained from the treated and the control groups of OE33, OE19 and FLO-1 cells  are conserved family of proteins and are multifunctional. Downregulation of these essential molecules by resveratrol could lead to loss of cell viability and have an effect on molecular entities of the esophageal cells, as observed in the molecular and cellular analysis that we performed.X-ray repair cross-complementing protein 5 (XRCC5), also termed Ku80 is an essential protein involved in repairing DNA damage along the NHEJ pathway. Downregulation of this central entity in the present study in the three cell systems studied, could have a major impact on cell proliferation, apoptosis and inhibition of key regulatory pathways operating in esophageal cancers, which are known to be aggressive in nature.Keratin type II cytoskeletal 8 plays a major role as protector of epithelial cell integrity. Downregulation of this molecule by resveratrol in esophageal cell system in the present study could have significant effect on the cell viability that we observed in cellular level assessment.Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) are a family of proteins which are splice variants that are closely related, and therefore, they are treated together as hnRNP A2/B1. A decrease in this key entity in all the three esophageal adenocarcinoma cell systems that we observed in the present study could have a profound impact on key pathways in this type of aggressive cancer.BH3-interacting domain death agonist (BID) has been reported to be elevated in certain tumors and has also shown to be involved in alternate apoptotic pathways. Therefore, a decrease in BID by resveratrol could alleviate other alternate pathways that could potentially operate in evading apoptosis.Transcription factors, such as BTF3, play a central role in controlling the gene expression of cancer genes. Downregulation of this factor in the three esophageal cell lines that we observed by protein profiling could have a deleterious effect on esophageal cancer cells.Among the upregulated proteins by resveratrol in the present study, heat shock protein HSP 90-beta was one of the proteins identified. By binding to other key molecules, it is reported to cause apoptosis. Therefore, it could be playing a role in the induction of apoptosis that we observed in the assessment of molecular mechanism of action in the present study.Serpin H1, also termed HSP47, is known to be elevated in cancers and was one of the upregulated components in the present study. Interestingly, the Serpin family of proteins are assessed in improving DNA vaccines against cancers. An increase in this component could aid in increasing the efficacy of DNA vaccines against esophageal cancers and to pursue chemo-immunotherapy treatment.Heterogeneous nuclear ribonucleoprotein A1 (HnRNP A1) was also upregulated. This molecule belongs to the family of RNA binding proteins, also termed RBPs. An increase in this entity is reported in cancers.Anterior gradient protein 2 homolog was one among the upregulated moiety. This protein belongs to the protein disulfide isomerase family. One such protein disulfide isomerase termed PDIA3 has been reported in esophageal cancers and plays a multifaceted role in cancers.Cofilin was also one among the upregulated molecule identified by proteomic profiling. There are reports of it as a therapeutic target in the treatment of cancers.The overall data from cellular analysis, molecular mode of action assessment and proteomic profiling has shed light upon molecular and immunomodulatory signatures, which could be harnessed for developing efficacious chemo-immunotherapy as well as chemo-radiation treatments for adenocarcinoma of the esophagus—an aggressive form of cancer.In We have attempted to decipher the action of resveratrol on adenocarcinoma of the esophagus with the aim of identifying key novel targets by multi-level profiling to combat the malady. Our study was conducted in the esophageal cancer cell lines OE33, OE19 and FLO-1. In the absence of well-defined animal model system to study adenocarcinoma of the esophagus , we condStudies on the mechanism of action of resveratrol were performed by flow cytometry. Flow cytometric analysis has been reported to be highly sensitive ; furtherWe sought to elucidate in detail the mode of action of resveratrol by protein profiling. Two-dimensional gel electrophoresis and mass spectrometry revealed differentially regulated proteins. We selected some of them based on the fold changes in the levels of the proteins observed in the three esophageal adenocarcinoma cell systems studied. We have categorized them based on the cellular pathways they regulate.In the category of protein synthesis machinery, elongation factor 2 was downregulated in resveratrol-treated esophageal cells. This factor, known to control cell proliferation, was reported to be overexpressed in ovarian cancers , while iPeptidyl-prolyl cis-trans isomerase B (PPIases) are conserved family of proteins and are multifunctional. They control various key activities in the cell, such as protein folding, trafficking and as chaperones. They also belong to the category of ‘Immunophilins’, and are receptors for immunosuppressant molecules. Having chemotactic and inflammatory properties, they play a role as vehicles for eosinophils, basophils and monocytes . FurtherSimilarly, PIN1 is overexpressed in esophageal cancers and forms a prognostic marker and a novel therapeutic target for esophageal cancer . As thisX-ray repair cross-complementing protein 5 (XRCC5), also termed Ku80, is an essential protein involved in repairing DNA damage along the NHEJ pathway. It is involved in multiple pivotal pathways, such as cell proliferation, telomere maintenance, apoptosis and phosphorylation of various key cellular factors including HSP90 . In hepaMoreover, in Head and Neck cancer cell lines, the sensitivity to radiation was shown to be correlating with levels of Ku80 . It alsoKeratin type II cytoskeletal 8 plays a major role as protector of epithelial cell integrity. Most adenocarcinomas express cytoskeletal 8. If keratin expression is aberrant, it is known to lead to drug resistance. Keratins play a pivotal role in motility, growth and protein synthesis as well. As keratins play a major role in cancer, they could be considered not only as diagnostic and prognostic markers but as therapeutic targets as well . In esopHeterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) are a family of proteins which are splice variants that are closely related and differ only by 12 amino acids, and thus, they are treated together as hnRNP A2/B1. These are a group of proteins that bind RNA and are also termed RBP. They play a central role in controlling various signaling pathways, including transcription, DNA repair, mRNA metabolism, splicing, transport-RNA shuttling and modifications at the post transcriptional level, as well as being involved in the biogenesis of telomeres at the ends of DNA. Therefore, deregulation of these proteins would play a pivotal role in cell pathological conditions. In lung cancer such as NCSLC, upregulation of A2/B1 is reported . In factBH3-interacting domain death agonist (BID) has been reported by immunohistochemical studies to be elevated in certain tumors such as cancers of the prostrate, colon, brain tumors such as gliomas and in lymphomas. Interestingly, higher amounts were detected in advanced tumors of prostrate and lymphomas. It is envisaged that alternate apoptosis mechanisms might be contributed to high levels of BID . In our Control of gene expression by transcription factors plays a key role in neoplasia. Transcription factors, such as BTF3, are shown to control tumor-associated genes in pancreatic cancers . In coloAmong the upregulated proteins by resveratrol in the present study, heat shock protein HSP 90-beta was one of the proteins identified. HSP 90 is a cell chaperone playing an important role in cell survival and is a chemotherapeutic target for cancers. There are reports of its role as a proapoptotic molecule as well; it was found that in leukemic cells, HSP90 interacts with c-Jun N- terminal kinase (JNK) in lipid rafts when Edelfosine was administered as an antileukemic therapy to induce the apoptotic response . In factSerpin H1, also termed HSP47, is known to be elevated in cancers. We observed upregulation of this entity on resveratrol treatment in the three esophageal adenocarcinoma cell lines studied. Interestingly, the mouse homolog Spi6 of human serpin P19 has been reported to have an effect on CD8-T cell responses by having an effect on the DC antigen-presenting cell’s viability . NotablyHeterogeneous nuclear ribonucleoprotein A1 (HnRNP A1) belongs to the family of RNA binding proteins also termed RBPs. HnRNP A1 in particular is shown to be involved in lung cancer proliferation  AlthoughAnterior gradient protein 2 homolog belongs to the protein disulfide isomerase family . One of Cofilin was also one among the entities upregulated by resveratrol. Overexpression of Cofilin is considered to lead to the invasion and metastasis in certain cancer cell lines ; howeverIn our present study, the observed differences in sensitivity to resveratrol could be due to the varied cancer driver gene status in the cell lines. Apart from cancer driver genes such as TP53 and Myc, the expression of the PSMD3 gene, which codes for the 19S component of the proteasomes, differs in the three cell lines . AdditioIn conclusion, the phenotypic outcome observed with resveratrol could be based on the stoichiometry of the fold changes per se of some of the pivotal entities identified in our study. Based on the proteomic analysis, DNA repair, DNA replication, transcription, cytoskeletal moieties, actin binding component and proliferation pathways were impacted. In the future, compounds that are synergistic with resveratrol could be pursued as well in a combinatorial approach. Importantly, proteomic profiling on resveratrol treatment has shed light on the pivotal cell signaling pathways and has highlighted molecular and immunomodulatory signatures such as Serpin (HSP47) and Anterior gradient protein 2- a protein disulfide isomerase with implications for chemo-immunotherapy as well as the development of anticancer vaccines for adenocarcinoma of the esophagus. Moreover, the downregulation of the pivotal entity Ku80 in the three-cell system studied could have a major impact in decreasing resistance to radiation therapy in adenocarcinomas of the esophagus, which are known to be highly aggressive in nature. Therefore, further studies are warranted with resveratrol as a treatment for esophageal adenocarcinomas.