NF-κB activation impairs somatic cell reprogramming in ageing
Authors: Alejandro De Los Angeles
José I Martín-Subero
Carlos López-Otín
Pablo Menéndez
José M P Freije
Clara Bueno
Clara Soria-Valles
George Q Daley
Ana Gutiérrez-Fernández
Fernando G Osorio
Journal: Nature Cell Biology (2015)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Ageing; somatic cell reprogramming; induced pluripotent stem cells (iPSCs); NF-κB activation; reprogramming repressor
Affiliations: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006-Oviedo, Spain., Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA., Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Faculty of Medicine, 08036 Barcelona, Spain., 1] Josep Carreras Leukemia Research Institute, Cell Therapy Program of the University of Barcelona, Faculty of Medicine, 08036 Barcelona, Spain [2] Institucio Catalana de Recerca i Estudis Avançats (ICREA), 08035 Barcelona, Spain., Departamento de Anatomía Patológica, Farmacología y Microbiología, Universitat de Barcelona, IDIBAPS, 08036 Barcelona, Spain., 1] Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA [3] Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, Cambridge, Massachusetts 02138, USA.

Abstract:  Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies. 

Retracted: Yes

PubPeer comments:
 When comparing figures in this article with the  corresponding raw data in the supplementary (western blots) I do not understand how they can possible correspond with the western blots presented in the figures. Figure 2a: the Lamin B1 whole blot in the supplementary is different from the two other blots (number of lanes and a blank lane in the midle). Figure 6: The IKFa has 9 lanes, the other blots have 8 lanes. The lanes used in figure 6 seem to have been cut from lane 5-7 for the actin and pATM blots, but for p-IKK2 lanes 6-8 seem to have been retrieved from. It is difficult to understand which number of lanes have been cut for the IKBa blot. Figure 7: the IKFa blot is different from the other blots (9 lanes (including one blank) vs 8 lanes for the others. It is also impossible to verify if the IKFBa blot is identical to the IKFBa blot in the supplementary. In addition it s hard to see how the NF-kB blot in the supplementary corresponds to NF-kB in figure 7. I will be grateful for any comments. ![file] ![file] ![file] , 
 The issues of WB images in this paper should not be ignored. Example, figure 7a, NF-kB blot does not match with raw data. The shape of bands is very different... ![file] , 
 Adding more issue in this topic. NF-kB blots in fig.3c never match with raw data. Shape, intensity and lane are totally different from the ones presented in main figure. The raw data does not say pBABE NGPS has strongest signal of NF-kB. The blots in raw data were never labled, so hard to identify which sample is. In any case, there is not any sample that shows outstanding intensity of NF-kB signal in raw data. Thus, presentation in fig.3c, NGPS cells has high level signal of NF-kB, is not valid. ![file] , 
 Another and similar issue found in supplementary figure 8. NF-kB bands from figure are very hard to compare with raw data in Soria-Valles et al. Shape and intensity differ a lot. ![file] , 
 How could the authors justify their quantification method and statistics of Tra1-60+ colonies? - I cannot understand too small error bars between samples from reprogramming experiments. Weird that Reprogramming efficiency is still variable nowdays... Are they from three biological replicates, or technical replicates? Quantified in blind mammer? - Here is a representation of their weird quantification. I do not see similar number of Tra1-60+ colonies between IKKi and IKK2-KI in figure 6d. ![file] , 
 #6 And other samples through IKKi to ATMi indeed look very different colony number, but bar graphs are so similar with minimal error bars... , 
 Riddle of  raw data of supplementary figure 6a. Uncertain crop. ![file] , 
 #6 Indeed, it is really difficult to understand how the authors justified which are Tra-1-60+ colonies. They should disclose raw data of original counts and criteria they followed for counting. ![file] , 
 It s unclear why some commenters are re-posting images (as Imgur links) that are already present in previous comments. Comment #8 re-posts the image from #6 Comment #11 re-posts the image from #10 Why is this being done? It doesn t seem to be helping, clarifying, or adding anything. , 
 The unclear resolution of submitted original image of WB conceals precise comparison with each figure. , 
 Supplementary figure 5 b and e showed percentage of colonies, both described as similar as around 60%, but the images of colonies in b and e look very different from their description. Panel b has way more colonies than panel e. The actual count and bar graph do not match. ![file] , 
 None of their submitted original films of WB were labeled. Only retrospectively annotated for figure preparation, that allows possibility of calling any sample as they wanted. Even though they annotated molecular weight (KDa), I do not see molecular weight markers run on the whole membrane. ![file] , 
 #15 Can anyone tell how the authors managed quantification of their stainings? It is entirely unclear how they measured the percentage of positive cells per field of images. From methods section,  Cell senescence was assessed by detecting the senescence associated u03b2-galactosidase activity at pH6.0 (SA-u03b2-Gal) using a commercial kit (Cell Signaling). , 
 I am following the former comments on the paper, considering arguments around NF-kB that affecting the main message of this study. NF-kB blots (EMSA) in this paper were too much intensely developed and lost its resolution as you can see in raw data. Nevertheless, their main figures showed relatively cleaner blots that can not found in raw image. Because of NF-kB being main points of this study, I would say that the author should go back to check the original blots. However, even if it were done, the problem still remains that none of the blots were formally labeled, so they could claim any lane as their actual sample ... ![file] , 
 Representative pictures linked to the statistics are just that, representative pictures...they do not have to represent the exact average values indicated by the graphs...that s why stats are for... Moreover,  None of their submitted original films of WB were labeled. Only retrospectively annotated for figure preparation, that allows possibility of calling any sample as they wanted. Really? this is absolutely ridicolous....and applicable to 100% of scientific publications... , 
 The blots of p16 were flipped. The blots of lamin B1 seem to come from different membrane of unrelated samples. It is hard to presume which blots correspond to which sample, because 1) The order of sample differ between each blots even in the same set of experiment; 2) The orginal mambranes were not labeled. I would share the view of Purpuradusta Barbieri that  allows possibility of calling any sample as they wanted . ![file] , 
 #23 Just for the record, even if the authors wanted to, digital imaging systems do not allow to label any original  membrane (I guess that Rubrimonas Cliftonensis means film). , 
 This is not a very old paper. Authors could still have the original data of raw blots and colony count . Please post them (and in high resolution...). , 
 #19 Lack of clear and solid standard of colony countings yields inconsistent results and representation. , 
 #23 Weird... It is hard to see how they conducted experiments. Did they run all the samples same day to make corresponding raw data to fulfill demand of NCB? , 
 It is very concerning that each band were flipped, intensity of signal was artistically modulated, and came from random film. Rubrimonas Cliftonensis pointed the cherry-picking of bands from irrelevant films. , 
 Ok, extensive posts on this paper might be reconciled into two major issues. a. The blots are flipping and jumping aroud different films. And contrasts of bands are too much modified. This issue accounts for most half of the paper (figure 2a, 3c, 4g, 4i, 5f, 6b, 7a, 8b, s1e, s2b, s4c, s6a, s6c, s6e, s8a-e). b. The criteria of colony counting is unclear and subjective. This issue accounts for another half of the paper (figure 1b, 1d-g, 3c, 3e-g, 5a-b, 5d-f, 6a, 6c-d, 7b-c, s1g, s4a-b, s5b-c, s5e-f, s6b, and s6d). , 
 #30 And do not forget more crucial issue on this paper that blots of NF-kB do not match with corresponding raw data submitted by the authors, that had been discussed on earliest posts. That affects validity of central claims on this paper that NF-kB impairs reprogramming of aged cells. , 
 - The raw data of control beta actin bands are missing. - P-p53, p53 and p16 bands were flipped. - P21 bands seem to come from irrelevant membrane. Their layout and lanes never match with other samples. - There is some weird baked membrane (or black box) on p16 membrane. ![file] , 
 #32 Where are their beta-actin control bands coming from...? , 
 #20 Dalbergia Nitidula, I guess you are right. The raw blots were too much modulated to correlate with images in figures. Raw films were so black, and figures were too white! The submitted raw blots (supplementary figure 9) lacks molecular size markers in every single film, and constrast was adjusted too high. NCB has policy below to raw blots.  Positive and negative controls, as well as molecular size markers, should be included on each gel and blot  High-contrast gels and blots are discouraged, as overexposure may mask additional bands.  And the criteria of the authors do not meet with the policy... , 
 #35 Authors, please post raw blots to comply with NCB s policy. , 
 Another NCB policy regarding quantification of microscopic images was not followed in this paper. It has been unclear and unnamed how the authors processed software of image quantification that occupied half of their data (figure 1b, 1d-g, 3c, 3e-g, 5a-b, 5d-f, 6a, 6c-d, 7b-c, s1g, s4a-b, s5b-c, s5e-f, s6b, and s6d). Tetramerium Zeta pointed that intuitive counting skill ended up similar percentage in two different experiments, but obvioulsy looking very different... NCB policy;  Processing software should be named and manipulations indicated (such as type of deconvolution, three-dimensional reconstructions, surface and volume rendering,  gamma changes, filtering, thresholding and projection). ![file] , 
 #32 I need multiple posts to address elusive bands appearing from nowhere. In addition to missing b-actin bands in supplementary figure 6a, there are multiple cases where b-actin or Oct1 (control of EMSA assay) are missing. First, Figure 4i. H3K79Me2 bands are flipped. B-actin bands are missing from submitted raw blots from authors. ![file] , 
 Figure 5f. ATM bands are distorted. b-actin bands are missing from raw blots submitted by authors. ![file] , 
 Figure 7a. Never specified which Oct1 bands are used. 4 out of 15 lanes. P-ATM bands are flipped. B-actin bands are missing from raw blots submitted by authors. ![file] , 
 Supplementary figure 6e. NF-kB bands do not match. Oct1 (control) bands are missing from raw blots submitted by authors. ![file] , 
 Supplementary figure 8. Part 1 of 2. Oct1 (control) bands are missing from raw blots submitted by authors. 9 lanes shown in the panel, but only 4 can be found. ![file] , 
 Supplementary figure 8. Part 2 of 2. B-actin bands are missing from raw blots submitted by authors. ![file] , 
 Policy of Nature Cell Biology on gel data deposition -  Positive and negative controls, as well as molecular size markers, should be included on each gel and blot . The paper lacks both controls and molecular size markers. I wonder if the editor of Nature Cell Biology, Alexia-Ileana Zaromytidou, is aware of it? , 
 #10 Paraplanocera Oligoglena, can you specify what is your argument here? I do not think one can justify percentage of colonies per field by manual counting. , 
 #47 My point is that the authors cannot depend on subjective colony counting. Unless conveyed machine and software-based counting, how could you be confident of the resultsu2026? I carefully looked for methods section in this paper, but never found any specific description regarding how the authors did counting. Indeed, counting from independent experiments did not correlate. Later posts argued that very different two samples ended up u201csameu201d number of colonies, supplementary figure 5 b and e (if machine-based count was done, it would never occurred). I also tried to do counting on their representative image on figure 5, and I have no idea how the authors could come to the exact number claimed in the bar graph (I saw apparently way more colonies in the image). Paraplanocera Oligoglena , 
 The earlier posts argued that NF-kB blotts did not match with deposited raw data from Soria-Valles et al. Adding to this discussion, I point out that number of blotts were different between figures and deposited raw data. In Supplementary figure 6 and 8, one lane is missing from their raw data. Moreover, shape of bands and order of lanes indicated in deposited films did not correlate with figures. They seems to randomly cropped unrelated films. ![file] , 
 The colony counting issue seems to be relatively overlooked compared with blot issues in this paper. But it should gather more attention. I do not understand how the authors managed to count them. I post image of supplimentary figure 5, adapted from previous posts. Please let me know if one can compare 2 different top panels on the image (b and e) and can tell me if they actually have the same number of colonies... NCB policy;  Processing software should be named and manipulations indicated (such as type of deconvolution, three-dimensional reconstructions, surface and volume rendering,  gamma changes, filtering, thresholding and projection). ![file] , 
 Control Oct-1 bands came from scratch. Figure 7 shows 4 lanes of NF-kB and corresponding Oct-1 bands. However, Oct-1 bands were selected from irrelevant film with multiple lanes. Unspecified which lanes were chosen. Moreover, this Oct-1 film was serving as a stock of control bands in several different figures around the paper. ![file] , 
 Figure 7 https: imgur.com a B94jiKR , 
 #52 I cannot spot NF-kB and Oct-1 lanes in figure 7a to the original films as slipped into the supplement. Indeed, there is a significant discrepancy both in bands width and shape. , 
 #52 The policy of Nature Cell Biology regarding immunoblot.  Loading controls (e.g. GAPDH, actin) must be run on the same blot. As pointed out by Volvatella Angeliniana, loading control (Oct-1) and sample (NF-kB) are not on the same blot (figure 7a). , 
 Oct-1 bands were randomely chosen from one film, and slipped into 3 independent experiments (figures 3c, 6b, 7a). A stock of control bands on donor film were repeatedly used in irrelevant samples and experiments throughout entire paper. ![file] , 
 In supplementary figure 8 c and d, Oct-1 bands were chosend from one film, and slipped into 2 irrelevant experiments. It is against the policy of Nature Cell Biology  Loading controls (e.g. GAPDH, actin) must be run on the same blot. Moereover, This Oct-1 film as well ther other one were used as a stock of bands slipped into irrelevant experiments through out paper. ![file] , 
 There is no molecular size markers in any immunoblots even in supplimentary figures where authors depositted raw blots. I remind policy of Nat Cell Biol.  Positive and negative controls, as well as molecular size markers, should be included on each gel and blotu2014either in the main figure or an expanded data supplementary figure. , 
 #56 NF-kB bands came from scratch. Number of lanes and shapes of bands do not match with deposited raw blots. As noted before, corresponding control bands were chosen from irrelevant pile of Oct-1 bands. ![file] , 
 #57 In addition to Oct-1, NF-kB bands and lanes do not match with original blots. 2 lanes were randomely chosen from film and slipped into independent experiments respectively. ![file] , 
 #60 What are middle blank bands in NF-kB blots in supplementary figure 8c, d? There are 2 lanes not annotated, and unrelated with control band blots. , 
 #63 No idea. There is no description about the blank lanes. There seems to be some bands of equivalant size as NF-kB. Wondering those bands were used somewhere in the paper... , 
 Figure 3c. There are some smears on the WB film of P-IKK2. They seems to be certain blots such as Nothern blot of unrelated samples. It is difficult to understand how WB and Nothern blots can be on the same film. And there is no molecular size marker. How the authors tell the size of bands? ![file] , 
 #65 Hibiscus Lunariifolius, Could you tell me how you can say the smear below the WB film are actually Nothern blots? It could be just some immunoblots of unrelated samples. , 
 Figure 3c and 6b. The complete lack of molecular size marker makes it difficult to tell if the entire film was submitted for deposition. The size of P-IKK2 and IKBA implies that they were from independent film. Just bands were pasted to represent entire film. ![file] , 
 Cell senescence assay in supplementary figure 5. Both samples A and B have very similar number of senescent cells under microscopic images. But the cell count says one is 5% (A) and the other is 60% (B). How did the authors count the cells? I could not find the quantification method in the paper.  Cell senescence was assessed by detecting the senescence associated u03b2-galactosidase activity at pH6.0 (SA-u03b2-Gal) using a commercial kit (Cell Signaling). ![file] , 
 Figure 6d. Colony number between IKKi and IKK2-KI has no difference according to bar graph. Though I found that actual image of colonies very differ.IKK2-KI has apparently 2-fold more colonies. Despite, error bars are really tiny. ![file] , 
 #59 Figs 3c, 6b and 7a, NF-kB and Oct-1 are differentially contrasted. NF-kB, whose bands are originally very faint, was super contrasted to become thick bands. On the other hands, Oct-1 were not highly contrasted, though the shape of bands were quite distorted so that match with the size with NF-kB bands. , 
 #71 Here is the image of figure 6b. NF-kB bands were contrasted a lot. Oct-1 bands were distorted. They came from different membranes, and received very different contrast respectively. ![file] , 
 Duplicated images in figure 3 and figure 4. Beta-actin bands were copied and pasted. ![file] , 
 #78 As long as the data in Figures 3 and 4 came from the same gel, they could be relative to the same beta-actin control, and it would make sense to repeat that panel in both figures for clarity. Disclaimer: I haven t checked to see if this is the case. , 
 #79 Hoya Camphorifolia, Figures 3 and 4 came from different gel. beta-actin was duplicated and reused for both figures. ![file] , 
 #78 The beta-actin bands were triplicated in 3 figures. Figs. 3, 4 and supplimentary 6. ![file] , 
 #69 Supplementary figure 5 worth more attention. The actual images and bar graphs contradict. ![file] , 
 #83 Comprehensive view of 3 figures with raw films. - How come one single control serves over 7 blots. - Some of the samples were apparently coming from different gels. - Identical bands were detected on different blots. Pasted...? - Trace of cropping bands were detected. ![file] , 
 #85 They need to comply with the policy of NCB journal to explain how they had counted cells.  Authors should list all image acquisition tools and image processing software packages used. Authors should document key image-gathering settings and processing manipulations in the Methods. The current method section has no description on this regard. , 
 #60 This mismatch of bands with original blots is an allegation against NCB polycy; The bands were too much overexposed and contrasted to be in shape, if the bands in original blots actually correpsond to the ones in figures.  High-contrast gels and blots are discouraged, as overexposure may mask additional bands. Authors should strive for exposures with gray backgrounds.  , 
 The quantification in figure 4i depends on irrelevant loading controls from unrelated samples. Even if the actin bands were to come from corresponding samples (unlikely though), the guideline of Nature Celll Biology says  Loading controls (e.g. GAPDH, actin) must be run on the same blot. ![file] , 
 This paper does not comply with NCB guideline that  Loading controls (e.g. GAPDH, actin) must be run on the same blot.  All the loading control were run on different blots as revealed in supplimentary figure 9, deposit of all the blots used in the paper. ![file] , 
 Figure 8b, H3K79me2 and b-actin bands came from different samples. The bands were cropped from different blots of apparently irrelevant lanes. ![file] , 
 Both rabbit (Lmnb1) and mouse (p21) primary abs were exposed on the same blot used in figure 2a and supplementary figure 6a. The whole blots submitted by authors seem to be actually patchwork of bands. ![file] , 
 #94 Also, in figure 2a, p16 and Lmnb1 came from different samples. The bands were cropped from different blots of apparently irrelevant lanes. What s more, p16 bands were flipped during cropping. ![file] , 
 High-contrast image revealed that NF-kB bands used in figure 7a are actually smear and artificial (indicated in blue rectangle). Real bands appear on above area (red rectangle). Refer blot B, below one, for actual size of NF-kB (red rectangle). ![file] , 
 Single blot in supplementary figure 1e had three lines of bands (top one is beta-actin, middle and bottom ones are unknown), but they do not match with lanes each other. Each line of bands came from irrelevant lanes. ![file] , 
 Both rabbit (IKBA) and mouse (p16) primary abs were exposed on the same blot used in figure 7a and supplementary figure 6a. The whole blots submitted by authors seem to be actually patchwork of bands. Indeed, there was a trace of band cropping around p16 bands. ![file] , 
 beta-actin bands for figure 3c actually came from irrelevant lanes. beta-actin bands do not match with sample lanes on the raw blot, apparently pasted from somewhere. ![file] , 
 figures 3c, 4i and supp. 6a. -uncertain bands and objects on blots -trace of crop in p16 lane -bands were collected from random blots for p-p53, p53, p21 and p16 -image duplications ![file] , 
 In figure 2a, p16 flipped the order of samples CTR and NGPS. ![file] , 
 Band flipping coincides with ageing... P-IKK2 and P-ATM blots were developed from the same gel, as can seen in the shape and spot at right upper corner of gel (white arrow). P-ATM was developed, and striped. Then, P-IKK2 was developed. Bands of P-ATM were flipped when panel figure 7a was prepared. ![file] , 
 #110 Another sample flip occured on the other half of the same gel (figure 6b). In addition, samples were shifted. P-IKK2 and P-ATM blots were developed from the same gel, as can seen in the shape of right upper corner of above gel. P-ATM was developed, and striped. Then, P-IKK2 was developed. Bands of P-ATM were shifted and flipped when panel figure 6b was prepared. ![file] , 
 Why is there no labelling (date, exposure conditions, antibodies, lane details) or even a ladder marked on any of the films themselves at all? This is something grilled into me when I learnt Western blotting so as not to muddle up blots or their orientation. I wonder how the researchers remembered what film was what, let alone what each lane was? , 
 It doesn t matter when you use collage type of data presentation. Remember this is not science but art production! , 
 #115 As long as they are using some unique system like wrapping blots with transparent rubber thingy and label on. I adapted previous comments there lack of molecular markers and sample labels causing issues. #68 molecular size is confused. ![file] #110 brought attention to this blot having sample flipping. ![file] , 
 ![file] , 
 b-actin loading control for panel figure 5f was totally missing from raw data shown in supplementary figure 9. As mentioned in earlier posts, there was no label, exposure time and markers on blots, so how the authors could orientate lanes? ![file] , 
 #100 raised a serious concern. The NF-kB bands represented from elderly human specimen were actually smear of blots managed with low exposure to make it look like bands. It Does affect the main claim of this paper that NF-kB activation impairs somatic cell reprograming in ageing. ![file] , 
 #121 In figure 7a, both NF-kB bands and its correponding mediators P-ATM and P-IKK2 were flipped and switched so that old-doner derived samples looked to have activation of NF-kB pathway. ![file] , 
 #120 The lack of labeling, markers, antibodies used, and exposure time concerns how these different blots were assembled to figures 3, 4 and supplementary 6. Bands were flipped, lanes were irrelevant and duplicated between independent figures. ![file] , 
 #119 Also, figure 4i uses bands from irrelevant blots to quantification. The case in figure 8b is more problematic, because it involves band flipping, so the claim of the figure turns to be actually oppisitte. ![file] , 
 #124 As pointed out by #119, sample flip happened in Zmpste24 knockout progeroid murine models in figure 8. Hypermethylation of histone in progeroid mouse, actually present in wild type. The observations in this paper, hyperactivation of both NF-kB and DOT1L in ageing, were due to sample flipping. Therefore, the role of both NF-kB pathway and DOT1L in ageing become obscure. ![file] , 
 #96 I thought that two or more western blots photographed together on one film. That explains why blots of different primary abs appear on one film. To check this, I changed contrast of this original blot corresponding to figure 2a. As you see, there is no margin of different membranes on the film. So I would say those bands on the film came from single blot with one type of primary ab. As molecular marker and label are absent, I can not be sure what those bands are. ![file] , 
 In supplementary figure 1e, samples 10-11 were missing from BAF blot. Lanes 8-9 of BAF and b-actin blots did not seem to come from the same gel. Again, lack of molecular marker and label leaves the identity of bands with dismay. ![file] , 
 #121 Size of NF-kB was not consistent between figures 3c, 6b, and 7a. Which bands of NF-kB are right? ![file] , 
 Not only WB band issues, but also their colony counting is cryptic and worth more attention. Their represent images and bar graphs do not match. This is something grilled into me when Method section did not describe how the authors quantified images. This paper depends on colony counting a lot. Figures 1b, 1d-g, 3c, 3e-g, 5a-b, 5d-f, 6a, 6c-d, 7b-c, s1g, s4a-b, s5b-c, s5e-f, s6b, and s6d. ![file] , 
 Figures 3c, 6b, 7a. https: imgur.com a VrBaHhd , 
 #132 More prominent example can be found in Figure 6. Two independent samples, one had very less and the other had much more number of colonies in the original images, had alsmot identical bar with tiny error. p=0.006, n=3. Even if the other two experiments had equal number of colonies between samples, p value cannot be such low as 0.006. ![file] , 
 Supplelentary figure 6c has several issues. - pBABE p16 band was highly contrasted to become thicker, although its corresponding IkB-SR p16 band on the same lane was not. - the pattern of 3 black spots at the right corner of IkB-SR p16 band differ between figure and original data. Upper left spot nearest to band is thickest in the figure, but lower right one is thickest in the original data. - b-actin bands are different from raw data. ![file] , 
 #137 I agree that colony countings are too subjective and lack consistency unless employed machine-based countings. The earlier posts focused on the issues in colony countings in this paper. Also, I would like to point out that too tiny error bars throughout this paper is something grilling into me. ![file] , 
 Figure 7f tried to show that expression of NANOG was reduced in aged fibroblasts. However, human fibroblasts do not express NANOG regardless of age (Takahashi, 2007 Cell. PMID 18035408). Actually, another figure (supplementary 2) in this paper clearly showed lack of expression of NANOG in human fibroblasts. ![file] , 
 #137 Following the author s description of statistical analysis, two-tailed Studentu2019s t-test was done with n=3. Assume IKKi colonies = 15, 16, 17; IKK2-KI colonies = 16, 18, 20. The most favorable numbers for authors that I could estimate from their represent images and bar graphs in figure 6b. t=test will be p= 0.07418. Still never be p=0.006 that authors reported in the paper. Curious if source data is available. , 
 The same protein BAF showed different number of bands between figures. ![file] , 
 Supplementary figure 6c https: imgur.com a rjvo9kR , 
 Figure 7 and supplementary figure 2. https: imgur.com a YdHOrYC , 
 Supplementary figure 1e. https: imgur.com a inDyprb , 
 #141 The issue of colony counting and statistics all came from lack of source data. Actually, NPG has a policy to deposit source data used for any statistical analysis. Example, another NCB paper of the same year (2015) did deposit source data. https: www.nature.com articles ncb3073#supplementary-information , 
 #138 Really weird. Only  partial change of contrast could explain this difference of spots. I do not know if partial change of contrast is allowed in any journal including NCB. ![file] , 
 #131 NF-kB activation is the central part of this study. The obscure identity of bands could carry off the conclusion of the paper. , 
 critics to this paper were covered below. https: forbetterscience.com 2018 08 24 how-lopez-otin-et-al-mocked-raw-data-policy-at-nature-cell-biology , 
 #138 In addition, the right two lanes of p16 had double bands with similar density, which was not consistent to the other left two lanes as well as any of other p16 lanes shown in supplementary figure 9. Again lack of precise molecular marker and label of abs, samples and exposure times makes it is hard to tell teh identity of bands represented in figures. , 
 #139 The Methods section described in the paper. The colony counting did noy exploit machine counting, rendering subjective results as discussed in earlier posts.  Reprogramming eufb03ciency. For quantification of iPSC reprogramming eufb03ciency, retroviral transduction was carried out in parallel infections containing all of the retroviruses used for reprogramming plus a tomato-expressing retrovirus. The total number of human iPSC colonies was counted after immunostaining with Tra-1-60 specific antibody as previously described 36, or by detection of alkaline phosphatase activity in murine iPSCs (AP detection kit, Sigma) following the manufactureru2019s instructions. , 
 #140 Same example in figure 6. Expression of NANOG in adult human fibroblasts was detected, which is contradicting with their own supplementary data. ![file] , 
 Figure 6 https: imgur.com a IxwbEkl , 
 Thanks earlier posts with eagle eyes. The inconsistency of  NF-kB bands throughout the paper seriously hurts the main claim of this study. ![file] ![file] , 
 #155 I compared NF-kB bands used in entire paper. The ones of figure 7a (red arrow) were apparently smear at lower part of blots if the other bands (blue arrow) were to be correct. Thanks for detecting this issue. ![file] , 
 Figures 3, 6, and 7. https: imgur.com a HYB5oXc , 
 #153 Expression of NANOG in adult fibroblasts was documented in figures 6 and 7 in this paper. But it contradicts with supplementary figure 2 as well as Shinya Yamanaka s Nobel prize-winning paper (2007) that established human iPS cells.![file] , 
 #148 p16 bands of supplementary figure 6c underwent unequal image adjustment so that some part of the blot get more intensified. ![file] , 
 Another example of NANOG expression in human adult fibroblasts. In the same figure (supplementary figure 2), data are contradictory between panel b and f. ![file] , 
 Figures 6, 7 and supplementary figure 2. https: imgur.com a HXmwWp8 , 
 Supplementary figure 6c https: imgur.com a xfrh7OH , 
 Supplementary figure 2. https: imgur.com a OYsvJ8j , 
 NF-kB bands do not match with raw data in supplementary figure 8c. The expression of wild-type BAF actually provoked NF-kB according to raw data. However, paper reads  the expression of wild-type BAF prevented ATM and NF-kB activation ![file] , 
 Supplementary figure 8c. https: imgur.com a 9cJyK53 , 
 What is statistics...? ![file] , 
 Editorial comment was published from Nature Cell Biology. 04 October 2018 Editor s Note: We would like to alert readers that the reliability of data presented in this manuscript has been the subject of criticisms, which we are currently considering. We will publish an update once our investigation is complete. https: www.nature.com articles ncb3207#change-history , 
 Too late and too hidden! With the long list of serious problems with this article an Expression of concern should be the right way to warn the readers. As it is now