Revision Checklist for Science Signaling Research Manuscripts: Data Requirements and Style Guidelines

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1 Revision Checklist for Science Signaling Research Manuscripts: Data Requirements and Style Guidelines Further information can be found at: Please feel free to your editor with specific questions or if you have a question about manuscript preparation at initial submission. Scientific or stylistic problem Instructions for Revision Data and Analysis Standards - CRITICAL Data not shown All data must be shown. Please include data that is not shown in the Supplementary Materials, if the data are not critical to the interpretation of the findings, or remove any phrases or sentences that refer to data that is not in the manuscript. Be sure not to introduce references to data not in the manuscript or that have been previously published as you make revisions in response to the referees. Include all additional data either in the main figures and tables or in the Supplementary Materials. Be sure any added data is presented in order. Actions or notes to the Editor N values N values must be provided in the figure legends for every data panel (this includes the Supplementary Figures), and what N represents (for example, cells, blots, mice, or experiments) must be indicated. For data requiring quantification and statistical analysis, N must represent at least 3 independent biological replicates. For data that does not require quantification and statistical analysis, N=1 is not acceptable, and N of at least 3 is preferable. Representative data may be shown. Data added in response to the referee comments must be represent N of at least 2. If quantification and statistical analysis are required, N must represent at least 3 biological replicates. It is not scientifically valid to present standard error or deviation bars or perform statistical analysis on data from N < 3 biological replicates. N values and what they represent must be specified in the figure legend for the added data. Quantification and statistical analysis When making claims about changes (or lack of change), please show quantification of at least 3 independent assays and perform statistical analysis. Statistical analysis should be performed on amalgamated data from at least 3 independent experiments, not on the results of a single experiment. Western blots: Key statements about increases, decreases, or lack of changes in protein abundance, phosphorylation, association, and activation must be supported by quantification of data amalgamated from at least 3 Western blots (that represent independent biological replicates) and statistical analysis where appropriate. Showing the densitometry from a single Western blot is not acceptable; amalgamated data from at least 3 Western blots must be presented: The signal for the protein of interest must be normalized to that of a loading control when quantifying signals from lysates. The signal for a phosphorylated form of a protein must be normalized to that for the total abundance of that protein. When quantifying changes in protein-protein interactions, the signal for the immunoprecipitated protein must be normalized to that in the total lysate. Statistics tests Western blotting Please add statistical analysis to graphs wherever possible to support statements about increases, decreases, or lack of change in the variable being measured. After each P value, state the statistical method used. Please note that t-tests and ANOVAs may not be appropriate for data with a non-normal distribution (such as percentages or qrt-pcr data). t-tests may not be appropriate for multiple overlapping comparisons in an experiment. For multiple comparison tests, such as ANOVAs, please specify the post-test used to derive P values. For guidance on selecting an appropriate statistical test, please consult a statistician and include the correspondence with the statistician with the revision. Please do not cite previously published papers as evidence that the statistical tests used are appropriate for the analysis of your data. For all Western blotting data, including figure panels that do not require quantification: 1

2 Western blots for phosphorylated forms of a protein of interest must be accompanied by Western blots for the total protein, to show that changes or lack of changes in the phosphorylation of the protein of interest are not due to changes in the overall abundance of the protein. This requirement also applies to other posttranslational modifications. Each set of Western blotting data must include an input blot, such as for actin or tubulin. Input blots (not actin or tubulin) must be shown for all coimmunoprecipitation blots for both the protein being pulled down and the protein(s) of interest that coprecipitated. Non-contiguous lanes within or between blots should be made obvious (delineated with borders, if necessary) and stated as such in the legend. RNA interference Genotypes, genes, and mrnas Scale bars Graph units Availability of RNAi, microarray, mass spectrometry, or structure data Ideally, to control for off-target effects, knockdown experiments should be performed with more than one sirna or shrna OR rescue experiments should be performed in knockdown cells. As well, knockdown efficiency should be shown by Western blotting; this data can be presented in the Supplementary Materials. Please italicize the names of genotypes, genes, and mrnas, and use the proper gene names. Primary and precursor forms of mirnas are italicized (the functional, mature form is not). All microscopy images (including histology, light microscopy, and immunofluorescence) must have scale bars. A single bar is sufficient for a group of images in a panel if they are all the same scale; if not, please include the appropriate scale bar within each image. Note its size in the legend, not in the figure itself. Please include the units on every X and Y axis, as applicable. For quantifications based on densitometry, please use (a.u.) for arbitrary units. Critical: Per the Science journals policy on data availability, all RNAi screens, microarray, mass spectrometry, or structure data must be deposited in a publicly accessible repository prior to publication. Please provide the accession number in the paragraph after the last reference in the section called Data and materials availability. **Note: If your paper passes peer review, we will not edit the paper and schedule it for publication until we have this deposition information. Organization and Presentation Title and abstract Note that the title may not exceed 135 characters including spaces, and the abstract may not length exceed 250 words. Sections Please ensure the main text is organized under the following subheadings: Abstract Introduction Results Discussion Materials and Methods Supplementary Materials (see Supplementary Materials list further below) References and Notes Figure Legends For the Supplementary Materials, please provide a separate Word file that contains the figures and tables, each followed by its legend (including a title). The legends should be single-spaced and use Times New Roman as the font. Supplementary Materials list Each item in the Supplementary Materials must have a short title. Insert a list of these short titles between the Materials and Methods section and the References and Notes section. The list in the main text must match exactly to those in the Supplementary Materials, in the following order (not all elements may appear): Figures Tables 2

3 References (range of the reference numbers that are only cited in the Supplementary Materials) Excel tables Movies Software Data files Refer to any recently published Science Signaling Research Article or Resource for example lists or see the online revision instructions for authors. Subheadings Figure order Figure legend length and content Figure size & formatting Please ensure that all subheadings in the Results and Discussion sections are the same style: either phrases or full sentences. Each section (A, B, etc) of the main figures must be called out in the text in order. Please ensure these are in order, and if not, rearrange the figures and their legends or rephrase the text as needed. Be sure that any data added to the manuscript in response to reviewers comments is also presented in order in the Results before being mentioned in the Discussion. Figure and table legends should not exceed 200 words if possible. Legends should describe the figure or table, not recapitulate the Materials and Methods or include interpretation that belongs in the Results. Example: (A) Western blotting analysis for x, y, and z performed at the indicated time points on lysates from xx cells treated with xx for x hours [or simply treated as indicated if the figure labels are explanatory]. Blots are representative of x experiments. (B) qrt-pcr for the expression of x, y, and z relative to w in xx cells transfected with xx sirna. (C) Apoptosis assessed by [insert assay here] was quantified in xx cells treated with xx. Data in (B) and (C) are means ± [SD or SEM] from [3 or more] independent experiments. We recommend using Adobe Illustrator or a program that can generate vector-based images to prepare your figures. We prefer that figures be provided as PDFs with editable text. Please prepare your main figures at high resolution (300 dpi minimum for gray scale and color; 1200 for line art), at 3.5, 5.0, 6.125, or 7.3 inches wide. Use the smallest of these sizes that retains legibility. For figure labels, please use Helvetica between 6 and 9 point in size; do not use bold for any labels within the figures. For panel labels, please use 10 point bold. Please try to leave some room at least in which to start the legend on the same page, and please left-align the panels; the legend may start in any corner white space. Across the figures: Bands in Western blots should be similar in width. Bars in all bar graphs should be a similar, narrow width, and graphs should occupy a similar sized area. Images should be a similar size (or as appropriate to see the necessary detail). Please make graphs and images small but retain label legibility. For an example of a paper with wellprepared figures, please see Figure labels Paragraph after last All language guidelines further detailed below apply to the figure labels as well. Most commonly: Do not use the word level (or levels), and do not use expression unless referring to genes or mrnas. If referring to mrnas, genes, or genotypes, use the proper gene name and italicize it. Ensure that gene and protein names conform to the appropriate species nomenclature. Avoid noting N.S. for non-significance within the figure unless the complexity warrants it; instead, indicate this and the test used to assess it in the legend. Avoid using anti-xx or α-xx to denote antibodies in the labels of Western blots (just label it with the protein name). The paragraph after the last reference should contain the following sections: 3

4 reference Acknowledgments, Funding Sources, Author Contributions (all authors must be mentioned), Competing Interests, and Data and Materials Availability (such as MTAs and accession numbers for data in public repositories). For example: Acknowledgments: We thank K.W. (AAAS) for providing the plasmids. We thank A.S. and L.T.M.F. for expert technical assistance. Funding: This work was supported by grants from AAAS (#88888 to N.R.G. and #77777 to J.F.F.). Author contributions: N.R.G. designed the project; A.V. and W.W. performed experiments; S.P. analyzed the mass spectrometry data; and L.K.F. and J.F.F. wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD The plasmids require a material transfer agreement from AAAS, U.S.A. References and methods in the Supplementary Materials There must be a single reference list and Materials and Methods for the entire manuscript. (Exceptions for Materials and Methods in the Supplementary Materials may include detailed computational methodology.) Any reference not cited in the main text should be added to the end of the main text references and numbered sequentially in the main text reference list and then referred by those numbers in the Supplementary Materials. Language and Stylistic Guidelines Use of tenses Please use the past tense to describe the results in your manuscript and the present tense to describe previously published results. Significant Slashes Amino acid callouts We reserve the use of significant for describing results of statistical analysis. Do not use it as a replacement for important or substantial. If you have used it to describe results that have not been analyzed by statistical tests, please use an appropriate statistical test to support claims of significant differences. Per style, we try to avoid the use of the slash due to its ambiguity. Please use and or or instead of a slash where appropriate, or define terms that will include a slash, such as protein names or complexes. For example, ERK1/2 should be defined as extracellular signalregulated kinase 1 and 2 (ERK1/2). To refer to single amino acid residues, please use the 3 letter abbreviation and superscript the residue number (for example, Ser 25 ); single letter abbreviations book-ending a normalscripted residue are used when describing amino acid mutants (for example, S25A). Express We use express and its related words to refer to (1) gene transcriptional events or (2) transgenic plants or animals or to transfected cells. It should not be used to describe the presence or absence, abundance, distribution, or location of a signaling molecule. Levels Up and down regulate Acronyms Antibodies In vivo, in vitro, in We use levels to indicate hierarchy (such as at the transcriptional level ) and not to indicate amount, abundance, or concentration. Per style, we avoid using imprecise terms, such as up-regulated, down-regulated, controlled, regulated, and modulated where possible. Please change up-regulated to increased abundance, increased activity, or whatever is appropriate and use similar language for down-regulated. Please change regulates to inhibits, attenuates, or reduces or stimulates, enhances, or promotes, as appropriate. Please define all acronyms the first time that they are used in the Abstract and the first time that they are used in the main text. Per style we do not refer to antibodies as anti-xxx. Please change in the text to an antibody recognizing or an antibody against or an antibody specific for. If anti-xxx is used in the figure labels, please define it in the legend. Per style, we do not italicize in vivo, in vitro, ex vivo, in silico, or in situ, but we do italicize 4

5 situ Other style guidelines et al. Please avoid claims of novelty or being first. Please do not start sentences with Importantly or Interestingly. Please avoid hyperbole and delete dramatically wherever you have used it. Our style is to use wild-type in the main text and WT in the figures. Note that it is not our style to use via, e.g., or i.e.. Please delete and rephrase wherever you have used these terms. Note that it is not our style to use elevated as a synonym for increased. Please rephrase. We avoid the use of recent and similar words and presenting previously published results as a historical perspective. Note that it is not our style to start sentences with As shown in Fig. 1B. Please revise throughout the manuscript. 5