#1
Reply to “Unregistered Submission: ( April 30th, 2014 6:46am UTC )” and to “Peer 1: ( February 1st, 2015 12:36am UTC )”.
Thank you for your remarks on some Figures of our paper that give us the opportunity to clarify some points. We would like to underline that we have been in contact with the Journal that chose not to pursue the matter.
1. Comments regarding multiple panels.
1.1 Regarding the concern that some panels are derived from different gels run at different times, we acknowledge that this is the case. As far as we remember, at the time we submitted the manuscript (2004), it was not forbidden to combine different images in a single larger panel within a figure. In the majority of the cases it was impossible to run in a single large gel kitetic assays performed using cell extracts obtained under different experimental conditions. Further, with the aim to provide the readership with high quality, logical, and consequential figures, we have often utilized images from different gels run at different times. Conversely, the kinetic assays related to the inherently stable GAPDH RNA (negative control) were, in many cases, run together as “final” assays when we already had an idea on how to organize a panel for a figure (Points related to Figures 1D, 1E, 2A, 3A, 3B, S3).
We are aware that now major scientific journals require that spliced gels are market by lines and we were ready to include separating markss in our Figures if requested by the Journal. The Journal chose not to pursue the matter.
1.2. Since many concerns have been generated by gels representing the decay kinetics of GAPDH mRNA, we would like, first, to clarify the significance of the utilization of GAPDH in our in vitro RNA degradation assays. GAPDH RNA decay patterns presented in different figures of the paper are not intended to provide a normalization for gel loading. GAPDH RNA decay profiles are representative of the consequences of incubation of distinct S100 cell extracts (either in the absence or in the presence of recombinant proteins) with a stable in vitro-synthesized radioactively-labeled RNA. Importantly, reactions including either unstable RNAs (such as myogenin, p21, etc.) or GAPDH RNAs are not run in the same gels. We performed at least three different independent experiments for each experimental conditions using either specific unstable RNAs or stable GAPDH RNA as substrates. Therefore, each gel showing the decay kinetic of a specific labile RNA upon incubation with a specific cell extract can, in principle, be presented in combination with each gel showing GAPDH RNA decay upon incubation with the same cell extract.
2. Comments to specific panels.
2.1 Bands of AREp21 RNA in lanes 13 and 14 appear to have more defined edges than other bands in the same panel since lanes 13 to 16 derive from an autoradiogram distinct from that presented in lanes 1-12 (please see the comment about autoradiogram splicing here above).
2.2 Comparison of bands in lanes 9 and 10 with bands in lanes 13 and 14. We must recognize that a careful analysis conducted on the original figures indicates a strong similarity between these bands. However, since it is clear that bands 9 to 16 of the AREcyclinD2 panel of Figure 1D belong to the same autoradiogram (there is no autoradiogram splicing) it is unlikely a fortuitous duplication. In any case, we have found in our records (dating more than ten years ago) at least three replicates for in vitro degradation experiments (all yielding superimposable results). Thus, we can provide the Journal with original autoradiograms if requested. Finally, I want to underline that part of the in vitro degradation experiments presented in Figure 1D were a confirmation of RT-qPCR analyses performed in the same cells under identical experimental conditions (shown in Figures 1A and 1B).
2.3 We do not detect any identity between bands 1 and 5 in AREcyclinD2 panel of Figure 1D. Let me also point to the fact that, in order to have consistency in the layout of the figures depicting RNA degradation assays, we ran the gels using always the same gel apparatus and the same comb. This resulted in bands presenting very similar shapes.
2.4. Panel showing IB anti-Tubulin in Figure 2A. The remark is correct. In order to maintain consistent the layout of the three panels, we placed the anti-Tubulin IB (added in the revised version of the Figure) in a larger frame (the reason for this choice was merely aesthetic). Please note that, conversely, we decided to maintain the original size of the anti-Tubulin immunoblot in the “sister” Figure S5 (U.V. crosslinking to AREp21) that was not destinated to the printed publication.
2.5 Figure 3A p21 panel. The effect is likely due to the fact that, at that time, the Polaroid prints were attached with adhesive tape to the laboratory notebooks. Therefore the scanner acquisition of the images resulted in unintended shades.
2.6 Beta2-MG panel in Figure 5E. Although lanes 3 and 4 look very similar, a careful inspection of the panel revealed some differnces. Also in this case, please note that we ran the agarose gels using always the same gel apparatus and the same comb. This resulted in bands displaying very similar shapes.
2.7 AREmyogenin panel in Figure 6C. Yes, we recognize that, in this case, the aestethic purpose was not fullfilled and band shapes and edges are not very similar one to the other.
2.8 Beta2-MG panel in Figure S7B. Even though we cannot detect the indicated vertical change, some imperfections in Polaroid prints might be due to either adhesive tape (see above) or to the acquisition procedure (scanning).
2.9 p21 panel in Figure S12. We recognize that the background of band in lane 1 appears darker that in other lanes. Please see point 2.8 here above for a possible explanation. Please let me note also that, besides the fact that several papers have reported the induction of p21 mRNA expression by DM in C2C12 cells, this evidence is presented also in Figures 1A and 3A of our paper.
Finally, please note that the Figures that raised your concerns have been generated and assembled in R. Gherzi’ s laboratory without involvement of the laboratories of the other two corresponding authors.