Tuesday, October 15, 2019

A turnip for the books

London police arrest man dressed as broccoli


Reuters reports, without offering any context, the fact that a man dressed as a giant broccoli was arrested yesterday in London. While being detained, he yells "give peas a chance."

Wherever a corporation is destroying the environment, Broccoli Man is there. Where there's muck there's brassica.

Friday, October 4, 2019

F-I-R-E-I-N-C-A-I-R-O

This post was earlier cross-posted at Leonid Schneider's site, hence the unfrivolous tone. The version there is improved by Leonid's editing, background details and frame-story. It is a sequel (or possibly prequel) to an earlier post.


The "Curate's Egg" joke has earned itself a special place in the history of integrity in scholarship, due to the outrage and calls for retraction, after the best-known version in a 1895 Punch turned out to have been plagiarised from a slightly earlier cartoon in a rival magazine. Not really true about the 'outrage', though please appreciate this Twiddle account, devoted to Victorian humour (the Golden Age of Dad Jokes). Here the yolk is the naive suggestion that in a homogeneous unitary product, only part might be decayed, leaving the remaining material perfectly palatable and reliable.



It may seem a stretch to get from there to Professor Gaetano Cairo's participation in PubPeer comment threads, where he has defended a half-dozen papers against criticism and hostile questioning. We should all admire his involvement, by the way, and hope that it inspires other researchers to do the same. But it is not really such a jump, for Cairo's courteous responses capture some of the curate's polyglossian positivity ('Polyglossian' is a portmanteau word I invented just now by combining 'Pangloss' with 'Pollyanna'). Their general theme is that one or two Figure components might have been taken by error from another context, or slightly fabricated by persons unknown, but the other results are above suspicion so the main conclusions of each paper remain unaltered.

For instance, "Loss of the von Hippel Lindau tumor suppressor disrupts iron homeostasis in renal carcinoma cells" (Alberghini et al. 2005) [5]. Here the objects of apologia are two loading controls (congratulations to those of you who had "Loading Control Library" in your Bingo cards!).



Murmuring 'Zoom... Enhance' like Deckard in Bladerunner, we tweak the contrast and find that the former control is an exercise in the artistic medium of decoupage.

Thank you for this remark. I would like to respectfully remark that the spot circled in red is invisible and the actin band in the yellow box would appear neatly cut if duplicated as suggested, whereas it appears rounded. The 18S are similar but also not so different from those in the adjacent lanes. Anyway, almost fifteen years after publication, the original films are no longer retrievable and thus it’s unfortunately impossible for us to evaluate them. We would like to remark that the potential image duplication, though inappropriate, does not affect the main results and conclusions of this work. In fact, the figures report a complementary experiment showing the well-known opposite regulation of IRPs and ferritins by iron and iron chelators.
Example (2) comes from "Doxorubicin paradoxically protects cardiomyocytes against iron-mediated toxicity" (Corna et al. 2004) [4]. To the casual eye, two or even three of the four loading-control lanes in Fig 3A are clones, with a few percentage variation in width, as can happen with identical triplets.


Thank you for this remark. The bands are very similar but also not so different from those in the adjacent lanes. It is actually hard for us to agree or disagree by inspection that the rRNA images were duplicated, as commented by Xanthorrhoea Gracilis. The paper is 15 years old and therefore, unfortunately, it’s impossible for us to retrieve and evaluate the original images of the ethidium bromide-stained rRNA bands used for this figure. In case of inadvertent error, we sincerely apologize, but we would like to remark that the hypothetical use of the same image for two different lanes, though inappropriate, does not affect the results and conclusions of this work.
The mystery was deepened by the emergence of the same rRNA band in Fig 3B of "Reactive oxygen species-independent apoptosis in doxorubicin-treated H9c2 cardiomyocytes" (Bernuzzi et al. 2009) [11] -- vertically compressed, and controlling across a quite different range of conditions.


It was accompanied there by another rRNA band in Fig 4A, in which the two right-hand lanes are novel (but identical) but the two left-hand lanes are ones we've already seen.

If arguendo we accept that the loading controls were assembled by cloning lanes within bands taken from elsewhere, then two corollaries follow: First, the lanes were not controlled against unintended fluctuations in the concentrations and quantities of lysate from each experimental condition. Second, whoever in the research team made up evidence to secure the desired outcome, what are the odds that they stopped after just one clue?

Crucially, the mystery manipulators may not have confined themselves to control bands. Go back to our first Instance of a problematic loading band, from Fig 4A of [5], which controlled across smeary clouds of Ft L protein (below, left). We have just met the same cloud-bank in [11], though flipped through 180°, recut in the manner of a purloined gemstone from six carats lanes down to four, and relabelled as 'HO-1'.


Other examples of repurposed bands can be found in this body of work... enough to start one wondering whether there was a single lab member in charge of pulling random blots out of the image archive when assembling a Figure, or did they share the task around on a roster? This next control appears twice in "Adenosine A(2)A receptor but not HIF-1 mediates Tyrosine hydroxylase induction in hypoxic PC12 cells" (Gammella, Cairo & Tacchini 2010) [12], as TFIID and as β-actin; in the latter manifestation it could easily be an X-ray of my index finger.


Another phalanx-bone had previously come to light in "Role of HIF-1 and NF-kappaB transcription factors in the modulation of transferrin receptor by inflammatory and anti-inflammatory signals" (Tacchini et al. 2008) [9].


[9] leads us to [8] and [13], "Adenosine A2a receptor-mediated, normoxic induction of HIF-1 through PKC and PI-3K-dependent pathways in macrophages" (De Ponti et al. 2007) and "Role of hypoxia-inducible factors in the dexrazoxane-mediated protection of cardiomyocytes from doxorubicin-induced toxicity" (Spagnuolo et al. 2011) -- the three papers being tightly intertwined despite their diverse topics. At left, [9] and [8] share a loading control which resembles a row of burning canoes.



In [9] the canoes are matched to a scene of full-rigged ships-of-the-line emerging through cannon smoke, in a little-known naval engagement from the Napoleonic Wars (above, right). In [13] the ships change their lane labels and lose one of their number, but acquire a new TFIID chaperone.

In less nautical imagery, two HIF-1 bands from Fig 1D of [8] and 1(a) of [13] are surprisingly similar after a horizontal flip:


Seriously, how does all this happen? We must consider the possibility that Prof. Cairo had only limited oversight of what was going on in the laboratory at the time of these papers.

Anyway, I haven't finished with [8]. Its Figures 6A and 7A feature a pair of Forensic Dental X-ray images, shared with Fig 5B of "Up regulation of IL-6 by ischemic preconditioning in normal and fatty rat livers" (Tacchini et al. 2006) [6]. One of those dental records is hard-working: it leads us back to 2010, with 3D of [12] (the neighbourhood of the phalangeal X-ray).


[6] rewards further attention. Within its Fig 5A, two "Oct-1" loading controls from Fig 2A are easily mistaken for a row of cat emojis, and cancel out surprisingly well when superimposed.


Their resemblance inspired 'Maianthemum Lichiangense' to draw four papers together into one diagram: Figs 1B from [8], 5A from [6], 2a from [9] and 3 from "Adenosine‐dependent activation of hypoxia‐inducible factor‐1 induces late preconditioning in liver cells" (Alchera et al. 2008) [10].


As the end of this post approaches, I belatedly realise that I never set out its purpose -- of providing some context for an earlier post. That one looked at the oeuvre of Prof. Alfonsina Desiderio, also of Milan University. This is either a sequel or a prequel. As Leonid noted in his Introduction to that post, there are historical links between the two research groups. It is not for me to wonder whether they speak of a broader research-culture malaise within Milan University.

There are also pictorial connections. For instance, this one, involving Figs 3(a) of "IRP1-independent alterations of cardiac iron metabolism in doxorubicin-treated mice" (Corna et al. 2006) [7] and 2C in a 2004 paper from Desiderio's group:


Now Prof Cairo did not find resemblance to be convincing. His frequent co-author Tacchini had collaborated with Desiderio in the latter paper, but not with his own team in the former, so the authorship lists of the two citations did not overlap:
Thank you for this remark. In reply to comments to a few other old papers I did not argue because the original data were no longer available for evaluation. However, in this case the comment of Pandanus Comatus about 18S RNA seems inappropriate because it regards two independent papers with no authors in common. I cannot see how the original image could have been duplicated.
It may be that a second image, shared between the respective Figs 2B and 3(a), is more convincing.


Finally, 'Macaranga Lowii' put together another sprawling diagram calling attention to similarities among a series of blots. The implication is that an original 10-lane band of S28 ribosomal RNA had been cut to various widths depending on the number of conditions retrospectively needing a loading control in each experiment.


Three of the five papers are currently relevant, though old enough to interest only historians... At the bottom of the chart is 'HIF-1-mediated activation of transferrin receptor gene transcription by iron chelation' (Bianchi, Tacchini & Cairo, 1999) [1]. At left, "Transferrin receptor induction by hypoxia" (Tacchini et al. 1999) [2]. At lower left, "Oxidative stress-mediated down-regulation of rat hydroxyacid oxidase 1" (Recalcati et al. 2003) [3]. There is some authorship overlap with the 2001 and 2002 papers from Desiderio's early work.


Conceding the possibility that a single band might have used more than once by mistake for loading-control purposes, Professor Cairo has pointed out that the lack of distinctiveness in the shapes of these Magritte-bilboquet blobs makes it difficult to be sure. Consulting the original data is impossible, given the largely antiquarian nature of these papers.
It is actually possible that we inadvertently duplicated the rRNA images, as commented by Streptomyces Avellaneus, though all these rRNA bands look alike. The J Biol Chem paper is 20 years old and the experiments were performed 1-2 years earlier, before the digital images era. Therefore, unfortunately, it’s impossible for us to retrieve and evaluate the original images of the ethidium bromide-stained rRNA bands used for this figure.
I agree that some of the proposed identities in the chart may be false positives, mistaken identities, But my general conclusions remain unaltered.

Sources:
[1]. "HIF-1-mediated activation of transferrin receptor gene transcription by iron chelation".
L. Bianchi, L. Tacchini, G. Cairo, Nucleic Acids Research (1999) doi: 10.1093/nar/27.21.4223 [PubPeer]

[2]. "Transferrin receptor induction by hypoxia. HIF-1-mediated transcriptional activation and cell-specific post-transcriptional regulation".
Lorenza Tacchini, Laura Bianchi, Aldo Bernelli-Zazzera, Gaetano Cairo, Journal of biological chemistry (1999) 10.1074/jbc.274.34.24142 [PubPeer]

[3]. "Oxidative stress-mediated down-regulation of rat hydroxyacid oxidase 1, a liver-specific peroxisomal enzyme"
Stefania Recalcati, Lorenza Tacchini, Alessandra Alberghini, Dario Conte, Gaetano Cairo, Hepatology (2003) doi: 10.1053/jhep.2003.50417 [PubPeer]

[4]. "Doxorubicin paradoxically protects cardiomyocytes against iron-mediated toxicity: role of reactive oxygen species and ferritin".
Gianfranca Corna, Paolo Santambrogio, Giorgio Minotti, Gaetano Cairo, Journal of Biological Chemistry (2004) doi: 10.1074/jbc.m310106200 [PubPeer]

[5]. "Loss of the von Hippel Lindau tumor suppressor disrupts iron homeostasis in renal carcinoma cells".
Alessandra Alberghini, Stefania Recalcati, Lorenza Tacchini, Paolo Santambrogio, Alessandro Campanella, Gaetano Cairo, Journal of Biological Chemistry (2005) doi: 10.1074/jbc.m500971200 [PubPeer].

[6]. "Up regulation of IL-6 by ischemic preconditioning in normal and fatty rat livers: association with reduction of oxidative stress"
Lorenza Tacchini, Lorenza Tacchini, Gaetano Cairo, Lorenza Tacchini, Gaetano Cairo, Cristina De Ponti, Marta Massip, Joan Rosellò-Catafau, Carmen Peralta, Free Radical Research (2006) doi: 10.1080/10715760600885432 [PubPeer]

[7]. "IRP1-independent alterations of cardiac iron metabolism in doxorubicin-treated mice"
Gianfranca Corna, Bruno Galy, Matthias W. Hentze, Gaetano Cairo, Journal of Molecular Medicine (2006) doi: 10.1007/s00109-006-0068-y [PubPeer]

[8]. "Adenosine A2a receptor-mediated, normoxic induction of HIF-1 through PKC and PI-3K-dependent pathways in macrophages".
Cristina De Ponti, Rita Carini, Elisa Alchera, Maria Paola Nitti, Massimo Locati, Emanuele Albano, Gaetano Cairo, Lorenza Tacchini, Journal of Leukocyte Biology (2007) doi: 10.1189/jlb.0107060 [PubPeer]

[9]. "Role of HIF-1 and NF-kappaB transcription factors in the modulation of transferrin receptor by inflammatory and anti-inflammatory signals".
Lorenza Tacchini, Elena Gammella, Cristina De Ponti, Stefania Recalcati, Gaetano Cairo, Journal of Biological Chemistry (2008) doi: 10.1074/jbc.m800365200 [PubPeer]

[10]. "Adenosine‐dependent activation of hypoxia‐inducible factor‐1 induces late preconditioning in liver cells".
Elisa Alchera, Lorenza Tacchini, Chiara Imarisio, Caterina Dal Ponte, Cristina De Ponti, Elena Gammella, Gaetano Cairo, Emanuele Albano, Rita Carini, Hepatology (2008) doi: 10.1002/hep.22249 [PubPeer]

[11]. "Reactive oxygen species-independent apoptosis in doxorubicin-treated H9c2 cardiomyocytes: role for heme oxygenase-1 down-modulation".
Francesca Bernuzzi, Stefania Recalcati, Alessandra Alberghini, Gaetano Cairo, Chemico-Biological Interactions (2009) doi: 10.1016/j.cbi.2008.09.012 [PubPeer]

[12]. "Adenosine A(2)A receptor but not HIF-1 mediates Tyrosine hydroxylase induction in hypoxic PC12 cells".
Elena Gammella, Gaetano Cairo, Lorenza Tacchini, Journal of Neuroscience Research (2010) doi: 10.1002/jnr.22366 [PubPeer]

[13]. "Role of hypoxia-inducible factors in the dexrazoxane-mediated protection of cardiomyocytes from doxorubicin-induced toxicity"
R D Spagnuolo, S Recalcati, L Tacchini, G Cairo, British Journal of Pharmacology (2011) doi: 10.1111/j.1476-5381.2011.01208.x [PubPeer]

No glass eyes? I am disconsolate*

https://www.theguardian.com/environment/2019/oct/04/monster-fatberg-autopsy-reveals-wet-wipes-bones-and-false-teeth


Everything made so much more sense when I realised that I am trapped inside a Jan Švankmajer animation.


* Not to be confused with Disconsulate, which is the diplomatic mission from Pandemonium, representing the interests of that Power. When they are not facilitating trade deals, the Disconsulate staff are busy providing assistance to those citizens who get themselves trapped within pentacles or otherwise entangled in the mortal domain (Hell is empty and all the devils are here).

Friday, September 20, 2019

A glitch in the Matrigel

This post was earlier cross-posted at Leonid Schneider's site, hence the nonfrivolity and Explaining Voice (and the images reduced to nigh-invisibility here). The version there is improved by Leonid's frame story, Tiger Lillies videos, t-shirt design and editing.

Recent news stories have shown that the way to buttress one's claims with evidence is to copy images and draw over them with a felt-tip pen. In this research tradition, the Milan University group of Desiderio and colleagues were ahead of their time.

So here is a Western Blot electrophoresis image, extant in versions with three, five and four columns, depending on how many lanes have been photocloned and then crudely retouched. They hail respectively from Maroni et al. (2011) (which I assume is the original), Bendinelli et al. (2013) and Maroni et al. (2015) - these are listed below as [6], [14], [9].



Suffice to say that when the two right-hand lanes of the five-lane version are superimposed, they cancel out more than one would expect, in the felt-pen-free sections.

Now Desiderio's laboratory group labour in the academic groves of molecular cell biology, and specifically, the subgrove devoted to metastatic breast cancer. They map out the network of metabolic proteins and signalling proteins involved when carcinoma cells go metastatic, or adapt to the hypoxic conditions inside a tumour. Their production of papers displays what one might call a kind of artistic unity, with images reappearing in diverse contexts across a decade, tying that oeuvre together in the manner of The Dude's rug... but also attracting a number of critical threads from the anonymous, incorrigible peanut gallery at Pubpeer.

That last sentence is there to foreshadow a "Loading-control Library" theme, but before diving into that, we have not finished with the "retouching" theme. Here's Figure 8(d) from [12] -- Maroni et al. (2014) if you like -- with details enlarged at the right:



The query from "Heterorhabditis Downes" on August 20 evoked a brief rebuttal from the last author on September 2:
The bands are similar but they are not the same.
Turning to Bendinelli et al. (2011) [7], the modifications to Figure 3(b) are more subtle, hinting of the airbrush more than the felt-pen. They might go unnoticed without contrast enhancement.


But all of Fig 3(b) deserves to be seen -- not just the single panel shown (twice) above. In the unavoidably over-sized diagram below, Fig 3(b) is right of centre. Left of centre is Fig 4A from Matteucci et al. (2013a) [10]; at top, Fig 2A from Bendinelli et al. (2014) [13]; and at bottom, Fig 9A [Hypoxia] from Maroni et al. (2011) [8]. Pay special attention to the 'vinculin' bands, which are the loading controls -- ensuring equal cell-lysate concentrations across the lanes -- made memorable in this case by the repeating 'sea-serpent' motif. The Desiderio group prefer vinculin for this purpose on account of its predictable good behaviour.


Evidently I am not alone in thinking "Ave Vinculin Invictus!" every time that useful, versatile protein resurfaces in a new context.


But we are not finished with [7]. 'Peer 1' wondered about the similarity in Figure 4D, between the 7th / 8th lanes of the 'vinculin' band and the 3rd / 4th lanes.


The same band had appeared slightly earlier in Previdi et al. (2010) [5], not endowed with duplicated lanes because that experiment only had six conditions for it to ensure equal loading across. See below, left. While below right, more airbrushing hides the join in Fig 4(b), between two copies of a single lane (serving to illustrate different treatments).



Peer 1's concerns with [7] reached the authors who sought to assuage them, not seeing any serious issue:
Figure 4B BBA 1813: We do not understand why the bands in the squares are evidenced because they are insignificant.
Figure 4D BBA 1813: It looks like a technical problem of the photographer in the preparation of the Figure also because there is a number “1” enclosed without significance.
Subsequent questions went unanswered, to the disappointment of anyone who hoped to learn the authors' current interpretation of Figure 1A. There, cultured carcinoma cells were enticed to migrate through a nutrient gel in a "Matrigel invasion assay", to test the migration-deterrent properties of different treatments:


Variously-coloured polygons have been added to helpfully highlight some of the duplicated or triplicated cells. The lower middle panel is particularly fertile. Further glitches in the Matrigel probably remain to be spotted (the Figure's resolution is lamentably low, making it hard to check the nuclear outlines to confirm any match), but after even one pair the horse is dead and will not benefit from further beatings.

Thus we encounter another of the unifying themes in the Desiderio oeuvre. Like a single small fish in a bottle of milk, the presence of a single duplicated cell in a Matrigel invasion assay is enough to erode one's faith in the veridical nature of the composition, and often there is more than one. From Matteucci et al. (2013b) [11], part of Fig 4E:


Figures 3B, 3D:


Also from 2013, here are the two right-hand panels from Fig 4E of [10]. The two left-hand panels can be omitted here as they duplicate Fig 3D from [11] (albeit rotated through 90° and labeled with different cell counts). We can also omit Fig 2D as its panels reappeared in 2015 as Fig 1C of [14].


Moving on to 2014, and [13], lovingly-curated collations of cells feature in Figures 1D, 2B, and 3A -- someone in the laboratory really enjoys composing them. Cells are spaced at implausibly-regular intervals, copied within but also between panels, increasing the resemblance to wallpaper. We only have space to admire 2B.


In contrast, the panels of Fig 1C from [14] remind me more of leopard-pattern faux-fur. If I were a student at the University of Milan, I would screen-print some of these collages onto t-shirts and sell them from a stall outside the Department of Biomedical Sciences for Health.


This whole line of enquiry was triggered by 'Actinopolyspora Biskrensis' when he scanned Fig 3D from Matteucci et al. (2018) [19] with the 'Forensically' duplicate-detection tool.



Evidently Forensically only reported photocloning within one single panel of Fig 3D. This could be taken as a challenge to see if the metal Tleilaxu unaided eye could do any better. Here are the two upper left-hand panels. Your move, Forensically.


A further clue that these images are not entirely kosher could be the sharp horizontal or vertical edges on some of the cells, marked by arrows. Also, the photoshopping of the cells would be vastly improved by turning their nuclei into googly eyes.

With the 'glitch in the Matrigel' theme finally out of the way (there are Invasion-assay images from 2008 and 2009 but the resolution is too low to make them worth scrutinising), we can return at last to the "Loading-control Library" theme. The Milan team accept that a loading control is a desirable adornment to an electrophoresis result, removing accidental or unconscious differences between experimental conditions and reassuring the reader that the researchers have not led themselves astray. But they are reluctant to waste time and laboratory resources creating them (vinculin might be a bugger to work with, for all I know), relying instead on a small library of equally-loaded bands, cutting off the desired length as if buying six or eight or 10 links of sausage from a butcher.

Did someone say "links of sausage"?

(see Fig 2 of [15])


(Fig 8C of [2]; then 3B; Fig 5B of [18]; Fig 8 of [12])


(Fig 4A of Matteucci, Bendinelli & Desiderio (2009) [4]; Fig 4B of [10]; Fig 3B of [13])

Fans of "Struwwelpeter" can think of them as collections of severed thumbs if they want an alternative metaphor.

We have already met the 'sea-serpent' motif, so now here is another oversized diagram, bringing together manifestations of a vinculin band most closely resembling a trail of burnt matches. The sightings span almost a decade -- from Ridolfi et al. (2008) [2], [4], through Bendinelli et al. (2009) [3], [12], Bendinelli et al. (2015) [15], to Matteucci et al. (2016) [17] and most recently Bendinelli et al. (2017) [18].


(see also Peer 1's version)

The band is flipped horizontally in one case, and often chopped into smaller sections as if visited by "The great, long, red-legged scissorman" and then reassembled (depending on the number of experimental lanes requiring calibration), but the shapes of the burnt matches are distinctive.

This is all leading up to the mother of all unavoidably oversized diagrams, in which 10 papers shared fragmentary glimpses of yet another library band. The creator (Clare Frances?) described it as "A slightly tangled web".



The authors accepted that the loading bands were erroneous but made no promise to correct them ¯\_(ツ)_/¯. For the burnt-match manifestations:
It looks like an inattention in the graphical assembling of the acquisitions, since the aspect of the vinculins is very similar. In any case the data are calculated on a triplicate.
An uncredited student was to blame for the Tangled Web.
A Phd who does not work any more in the group prepared these Figures.
Just saying, there would be no need to "assemble the acquisitions" if they hadn't been cut into small pieces. Things got so confusing that two different loading bands were used in Fig 3B of [13] and 7B of [17] -- sausages / severed thumbs in the former case -- to control for expression of the same Snail band.


Although the recurring themes within this corpus of work all have their illustrations now, I am unwilling to stop, for I do not know if I have conveyed its curious timelessness. The repurposing and re-splicing and retouching of images span the decade. One can begin anywhere in the network and finish anywhere by following the threads between Figures.

Begin, by way of example, with Matteucci, Bendinelli & Desiderio (2009) [4]. We pause to admire its most notable feature, Fig 2A -- an imposing palisade of immunoprecipitated protein fragments. Close, contrast-enhanced inspection reveals the internal repetitions and symmetries that betray its origins in Photoshop.



But do not let this distract us from Fig 5A, a short train of Wwox blobs. Later the train lost its three central carriages to become part of Fig 1B in Maroni et al. (2015) [14], with the train and caboose directly conjoined (in 2013, 1B was also Fig 2E in [10]).


[14] is a kind of hub station in this Metro-map of pictorial connections, linking in all directions. Consider Fig 6B, at left. It shares bands with Fig 2D from [9], but they flank other bands which are not shared, suggesting another visit from the Scissor-man.

Within [9], Fig 2D invites further comparison with Fig 4E. Lanes have been copied, flipped horizontally, and darkened as if by police constructing an identity line-up.



In keeping with this nascent tradition, Fig 2C (still at [9] by the way) resembles an identity line-up of suspension-bridge towers... "Do you recognise any of these as the Brooklyn Bridge you bought?" But few readers will be surprised to discover that Lane 7 is a horizontally-flipped, retouched version of Lane 4.

Then [9] leads back to [4], by way of a reused B23 loading control for Western blots of nucleus proteins, and a HDM2 band that appears to have been assembled from pieces of a TAZ band (flipping one piece and rotating another through 180°). But I don't have time to explain that because the Editor is waiting impatiently for this post. Nor is there time to explain the diagram below right, where [4] jumps back a decade, by way of Fig 1D and the lanes it shares with Fig 4B from Maroni et al. (2006) [1].



[1] in turn leads back to [3] from 2009, through forensic dental X-ray comparisons of their RNA blots.


For a change of pace, how about some mice, from [10]?


"Am I missing something with Figure 6A? It appears that for mouse Control 2, the Dorsal and Ventral views are the same at 15 and 22 days, although greater bioluminescence is shown at 22 days. For Control 1, the Dorsal views are different but the Ventral views are the same at 15 and 22 days... but again, greater bioluminescence is claimed for 22."
I have been neglecting [19], the most recent in this series. In Fig 1E, "Is there some reason why the upper and lower sections of each lane are identical?" In Fig 5B, "the upper and lower extensions of each lane display identical fine texture. Is this a common phenomenon?"



The sheer scale of the insouciant repurposing of lanes, bands and microphotographs is vertiginous. If the reader is exhausted, fair enough, but imagine how I feel after reading all those threads and trying to arrange just a fraction of the cut-and-pastes into some sort of semi-thematic sequence. I am trying to make excuses for the group... perhaps they are convinced that they have an accurate model of how the different proteins interact and cascade within cancer cells as oncogenesis progresses, so collecting visual data that would convince their audience is a mere formality. They might feel justified, then, in taking shortcuts in the production of that evidence.

I didn't say they were good excuses.

Sources:

[1]. "HGF induces CXCR4 and CXCL12-mediated tumor invasion through Ets1 and NF-kappaB", Paola Maroni , Paola Bendinelli , Emanuela Matteucci , Maria Alfonsina Desiderio (2006).
Carcinogenesis doi: 10.1093/carcin/bgl129 [PubPeer]

[2]. "Inhibitory effect of HGF on invasiveness of aggressive MDA-MB231 breast carcinoma cells, and role of HDACs", E Ridolfi, E Matteucci, P Maroni, M A Desiderio (2008).
British Journal of Cancer doi: 10.1038/sj.bjc.6604726 [PubPeer]

[3]. "NF-kappaB activation, dependent on acetylation/deacetylation, contributes to HIF-1 activity and migration of bone metastatic breast carcinoma cells", Paola Bendinelli , Emanuela Matteucci , Paola Maroni , Maria Alfonsina Desiderio (2009).
Molecular Cancer Research doi: 10.1158/1541-7786.mcr-08-0548 [PubPeer]

[4]. "Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells", Emanuela Matteucci , Paola Bendinelli , Maria Alfonsina Desiderio (2009).
Carcinogenesis doi: 10.1093/carcin/bgp080 [PubPeer]

[5]. "Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and beta-catenin/Wnt pathways", Sara Previdi , Paola Maroni , Emanuela Matteucci , Massimo Broggini , Paola Bendinelli , Maria Alfonsina Desiderio (2010).
European Journal of Cancer doi: 10.1016/j.ejca.2010.02.036 [PubPeer]

[6]. "Nuclear co-localization and functional interaction of COX-2 and HIF-1α characterize bone metastasis of human breast carcinoma". Paola Maroni , Emanuela Matteucci , Alessandro Luzzati , Giuseppe Perrucchini , Paola Bendinelli , Maria Alfonsina Desiderio (2011).
Breast Cancer Research & Treatment doi: 10.1007/s10549-010-1240-1 [PubPeer].

[7]. "Comparative role of acetylation along c-SRC/ETS1 signaling pathway in bone metastatic and invasive mammary cell phenotypes", Paola Bendinelli , Paola Maroni , Emanuela Matteucci , Maria Alfonsina Desiderio (2011).
Biochimica et Biophysica Acta doi: 10.1016/j.bbamcr.2011.06.004 [PubPeer]

[8]. "Nuclear co-localization and functional interaction of COX-2 and HIF-1α characterize bone metastasis of human breast carcinoma", Paola Maroni, Emanuela Matteucci, Alessandro Luzzati, Giuseppe Perrucchini, Paola Bendinelli, Maria Alfonsina Desiderio (2011).
Breast Cancer Research & Treatment doi: 10.1007/s10549-010-1240-1 [PubPeer]

[9]. "Hypoxia inducible factor-1 is activated by transcriptional co-activator with PDZ-binding motif (TAZ) versus WWdomain-containing oxidoreductase (WWOX) in hypoxic microenvironment of bone metastasis from breast cancer", Paola Bendinelli , Paola Maroni , Emanuela Matteucci , Alessandro Luzzati , Giuseppe Perrucchini , Maria Alfonsina Desiderio (2013).
European Journal of Cancer doi: 10.1016/j.ejca.2013.03.002 [PubPeer].

[10]. "Bone metastatic process of breast cancer involves methylation state affecting E-cadherin expression through TAZ and WWOX nuclear effectors", Emanuela Matteucci, Paola Maroni, Alessandro Luzzati, Giuseppe Perrucchini, Paola Bendinelli, Maria Alfonsina Desiderio (2013a).
European Journal of Cancer doi: 10.1016/j.ejca.2012.05.006 [PubPeer]

[11]. "Epigenetic control of endothelin-1 axis affects invasiveness of breast carcinoma cells with bone tropism", Emanuela Matteucci , Paola Maroni , Paola Bendinelli , Alessia Locatelli , Maria Alfonsina Desiderio (2013b).
Experimental Cell Research doi: 10.1016/j.yexcr.2013.04.022 [PubPeer]

[12]. "Osteolytic bone metastasis is hampered by impinging on the interplay among autophagy, anoikis and ossification", P Maroni , P Bendinelli , E Matteucci , A Locatelli , T Nakamura , G Scita , M A Desiderio (2014).
Cell Death & Disease doi: 10.1038/cddis.2013.465 [PubPeer]

[13]. "Microenvironmental stimuli affect Endothelin-1 signaling responsible for invasiveness and osteomimicry of bone metastasis from breast cancer", Paola Bendinelli, Paola Maroni, Emanuela Matteucci, Alessandro Luzzati, Giuseppe Perrucchini, Maria Alfonsina Desiderio (2014).
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research [PubPeer]

[14]. "Hypoxia induced E-cadherin involving regulators of Hippo pathway due to HIF-1α stabilization/nuclear translocation in bone metastasis from breast carcinoma", Paola Maroni , Emanuela Matteucci , Lorenzo Drago , Giuseppe Banfi , Paola Bendinelli , Maria Alfonsina Desiderio (2015).
Experimental Cell Research doi: 10.1016/j.yexcr.2014.10.004 [PubPeer].

[15]. "HGF and TGFβ1 differently influenced Wwox regulatory function on Twist program for mesenchymal-epithelial transition in bone metastatic versus parental breast carcinoma cells",
Paola Bendinelli, Paola Maroni, Emanuela Matteucci, Maria Alfonsina Desiderio (2015).
Molecular Cancer doi: 10.1186/s12943-015-0389-y [PubPeer]

[16]. "The Autophagic Process Occurs in Human Bone Metastasis and Implicates Molecular Mechanisms Differently Affected by Rab5a in the Early and Late Stages", Paola Maroni, Paola Bendinelli, Massimo Resnati, Emanuela Matteucci, Enrico Milan, Maria Alfonsina Desiderio (2016).
International Journal of Molecular Sciences doi: 10.3390/ijms17040443 [PubPeer]

[17]. "Coordinate regulation of microenvironmental stimuli and role of methylation in bone metastasis from breast carcinoma", Emanuela Matteucci , Paola Maroni , Andrea Disanza , Paola Bendinelli , Maria Alfonsina Desiderio (2016).
Biochimica et Biophysica Acta doi: 10.1016/j.bbamcr.2015.10.010 [PubPeer]

[18]. "Epigenetic regulation of HGF/Met receptor axis is critical for the outgrowth of bone metastasis from breast carcinoma", Paola Bendinelli, Paola Maroni, Emanuela Matteucci, Maria Alfonsina Desiderio (2017).
Cell death & disease doi:10.1038/cddis.2016.403 [PubPeer]

[19]. "Microenvironment Stimuli HGF and Hypoxia Differently Affected miR-125b and Ets-1 Function with Opposite Effects on the Invasiveness of Bone Metastatic Cells: A Comparison with Breast Carcinoma Cells", Emanuela Matteucci , Paola Maroni, Francesco Nicassio, Francesco Ghini, Paola Bendinelli, Maria Alfonsina Desiderio (2018).
International Journal of Molecular Sciences doi: 10.3390/ijms19010258 [PubPeer]

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Comment / Update #1:
Here’s yet another Desiderio Library Band, working its loading-control magic in six papers from 2008 to 2013:

Comment / Update #2:
However, in this case the comment of Pandanus Comatus about 18S RNA seems inappropriate because it regards two independent papers with no authors in common. I cannot see how the original image could have been duplicated. This casts some doubts about the accuracy of software-based image comparisons.”
Prof Cairo is indignant (or at least not entirely dignant) at the suggestion that Fig 2C in a 2004 paper by Tacchini and Desiderio could be in any way connected to Fig 3c in his own 2006 paper. His disavowal of Tacchini, and the argument that the resemblance must be merely a coincidence, are both weakened by the existence of a second parallelism between Figures 2A and 3b respectively.


Comment / Update #3:
[1] in turn leads back to [3] from 2009, through forensic dental X-ray comparisons of their RNA blots.

My favourite part of the Dental-X-ray RNA blots was a comment from Prof Desiderio, explaining that they were only for purposes of illustration… artists’ conceptions, as it were. She seemed to be genuinely puzzled that anyone could expect them to be factual.
Figure 2A Mol Cancer Res 7 and Carcinogenesis 28: The RNA 18S and 28S in the yellow square were only illustrative of RNA visualization.
The texture in the dark background of these blots (visible with increased lightness) is so similar from one lane to another, that they’re quite possibly all constructed by photocloning just one lane...


... and then retouching the detailed outlines of the “teeth”.


Comment / Update #4:
Bernelli Zazzera sought to extend his reach over public and charity money by recruiting the bigwig Alberto Mantovani to the University of Milan. Here is an example of the joint output, Tacchini et al 2003

That particular publication has other surprises: