The fatty acid oxidation product 15-A3t-isoprostane is a potent inhibitor of NFŒ∫B transcription and macrophage transformation
Authors: Ginger L Milne
Alessio Porta
Joshua D Brooks
Giuseppe Zanoni
BethAnn McLaughlin
Jocelyn R Howard
Erik S Musiek
Enrico M Brunoldi
Jason D Morrow
Jeannette N Stankowski
Giovanni Vidari
Tyler R Koestner
Journal: Journal of Neurochemistry (2011)
Discipline: NEUROSCIENCE & BEHAVIOR
Keywords: Fatty acids; eicosapentaenoic acid (EPA); oxidation products; cyclopentenone IsoP; 15-A(3t)-IsoP
Affiliations: Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8548, USA.

Abstract:  Fatty acids such as eicosapentaenoic acid (EPA) have been shown to be beneficial for neurological function and human health. It is widely thought that oxidation products of EPA are responsible for biological activity, although the specific EPA peroxidation product(s) which exert these responses have not yet been identified. In this work we provide the first evidence that the synthesized representative cyclopentenone IsoP, 15-A(3t)-IsoP, serves as a potent inhibitor of lipopolysaccharide-stimulated macrophage activation. The anti-inflammatory activities of 15-A(3t)-IsoP were observed in response not only to lipopolysaccharide, but also to tumor necrosis factor alpha and IL-1b stimulation. Subsequently, this response blocked the ability of these compounds to stimulate nuclear factor kappa b (NFŒ∫B) activation and production of proinflammatory cytokines. The bioactivity of 15-A(3t)-IsoP was shown to be dependent upon an unsaturated carbonyl residue which transiently adducts to free thiols. Site directed mutagenesis of the redox sensitive C179 site of the Ikappa kinase beta subunit, blocked the biological activity of 15-A(3t)-IsoP and NFŒ∫B activation. The vasoprotective potential of 15-A(3t)-IsoP was underscored by the ability of this compound to block oxidized lipid accumulation, a critical step in foam cell transformation and atherosclerotic plaque formation. Taken together, these are the first data identifying the biological activity of a specific product of EPA peroxidation, which is formed in abundance in vivo. The clear mechanism linking 15-A(3t)-IsoP to redox control of NFŒ∫B transcription, and the compound's ability to block foam cell transformation suggest that 15-A(3t)-IsoP provides a unique and potent tool to provide vaso- and cytoprotection under conditions of oxidative stress. 

Retracted: No

PubPeer comments:
 #2 It is certainly an impressive match!  , 
 #1 I am in the middle of a Title IX complaint with a faculty member who is being investigated for retaliation and harassment. He has clearly crossed a line and is now attempting to defame my student s work. I stand by these studies. , 
 This is the contributer of the #2 post. I have absolutely no connection to any of the authors of this study, or their research institutions. I m simply an observant reader of the literature that noticed duplication of figures between papers and some western blot splicing. Since the lead author is now aware of this problem they should post the original data and explain this figure duplication. These are clearly the same western blot images that have been electronically manipulated, re-used and labelled as different things. This is a big problem for these papers and needs to be addressed. , 
 I found this post extremely concerning, and though this was a long time ago, I dug through my old hard drive and found the original blots, which offer an rather pedestrian explanation. The A4-NP and A3-IsoP blots in the 2009 and 2011 paper are from the same experiment and are taken from the same membrane. The strip reblot did not always come out well, so I ran all the samples in the same order on another gel in parallel and blotted for ERK, used this as the loading control for all of the other blots (in retrospect not the best practice, but hardly fabrication). I treated several plates of RAW cells with LPS and either A4-IsoPs or A3-IsoPs and ran them on the sample western. Later, we decided not to include the A3-NP data in the 2009 paper, so I cut those lanes out and spliced the old gel together. This was not to obscure the truth, but rather to just remove intervening lanes. The original iNOS blot is here:  In the 2009 paper, I ran the same samples in parallel for COX-2, but I did not include the 5uM A4-NP lane (as I recall it had a bubble, can t find that blot), so I removed that lane (and used the ERK loading control blot as with the iNOS blot, with the 5uM lane removed). So the blots were spliced to remove intervening lanes, but as you can see form the original blot, it is a very nice blot and the data is quite clear and robust. The ERK blot did appear twice in the 2009 paper as the load control for both the iNOS and COX-2 blots, because it was. I left the lab in 2008 for residency, and Josh Brooks took up the A3-NP project. I had the left over lanes form this blot with the A3-NP samples and corresponding ERK load controls, which went with the original LPS control lane. I sent these and other data to Josh, to use as one of his replicates for quantification, but they ended up in the paper, perhaps unwittingly. As far as I know he replicated this blot several times himself, and the quantification data is in the paper. Thus, the problem is really just the  representative blot that was chosen to show, not the data itself. It s clear from my blots that A3-IsoP have similar inhibitory effects on iNOS as A4-NP. In writing this, I wish I had just run the samples over again in the correct order. But the data is still the data, and the raw blot shows that the effects are real and not the result of some surreptitious splicing of blots. Indeed, this was not a case in which 2 blots were spliced together to look like one, but rather where the same blots was cut to remove A3-IsoP lanes. As far as I know, the blots were not re-labelled as different things. , 
 #7 Much appreciated. Thanks for delving into your archives. , 
 Dr. Musiek, thank you for posting these original data and for the explanation. However, there are a few points that still require clarification. You state that the loading controls are duplicated in the JBC paper because they are for the same samples, but this does not appear to be correct according to the labelling of the blots. In panel B above the first ERK lane is the control for the LPS treated sample. However, in panel C the identical band is shown as the control for the non-LPS treated sample. Regarding the image duplication of data between the JBC and the JNC paper, this was not done by you, but by a colleague to whom you sent the data. However, notice that the incorrect lanes were used. According to the original data that you have posted (I commend you for maintaining your data archives), the lanes published in the JNC paper are not for the treatment with 15-A3t-IsoP at 5uM and 10uM as stated in the article, but rather they are the identical bands from the JBC paper which are A4-NP at 1uM and 10uM. Again, the same loading control is also recycled from the previous paper. It s clear from your comments that none of this was intended to be malicious or misleading, but common practice for western blotting is to run an independent loading control for each experiment. This is especially important for making quantitative estimates in western blotting, since the efficiency of protein transfer may vary from experiment to experiment. Also, identical images should not be duplicated between papers even if they are considered to be  representative blots, I understand this was not done by you but perhaps you could communicate with the other authors to issue a correction of the JNC paper to include the correct bands from your original blot for the 15-A3t-IsoP treated samples. , 
 Apparent recycling of western blot images from a previous paper. One of ERK loading control appears to be taken from a previous paper : https: www.ncbi.nlm.nih.gov pubmed 18490445 Also part of upper panel seems to be duplicated as well. Both seem to have been squished and stretched a little bit using photoshop, but the bands look remarkably similar. There s some issues with the other paper s figures also covered in a separate Pubpeer post.  , 
 Figure 2D Looks like a panel splice between lanes 2 and 3. This is problematic if the ERK panel below is the loading control, since they wouldn t be from the same blot. http: i.imgur.com GSGgXe7.jpg http: imgur.com GSGgXe7 , 
 It seems like a correction would be in order for this paper. Based on second-author Musiek s input in a previous post it s clear that the incorrect western blot was published in this paper (results are from a different drug treatment that the one stated in the paper). Since the correct data exists (see post#7) this would be a great opportunity to correct the literature and provide the journal with the correct data. ,