Efforts to Improve Transparency at the JBC Roger J. Colbran Associate Editor Credit: Amanda Fosang, Associate Editor

Why Improve Transparency? Irreproducibility of pre-clinical studies Negative impact on political and public will to fund research…...

Reason for irreproducibility Poor methodological descriptions Reagents not described adequately and/or validated – antibodies, siRNAs, small molecules Inappropriate data interpretation

NIH Principles and Guidelines Rigorous statistical analysis Transparency in reporting Establish guidelines for: Image based data Western blots Descriptions of biological materials Antibodies: source, dilutions, validation Cell lines: source, authentication, mycoplasma Animals: species, source, strain, sex, age

Efforts Toward a Solution…... Improve transparency of JBC papers by: 1.Improving description of methods, including reagents 2.Clearly defining reproducibility (technical and biological replicates)

1.Experimental uncertainty, statistics 2.Graphical presentation of data 3.Western blots – presentation and quantitation JBC Policies and Guidelines

1. Experimental uncertainty, statistics NIH: Require full reporting of statistics, including tests used, exact value of N, dispersion/precision (e.g., mean, median, SD, SEM, confidence intervals). Inclusion and exclusion criteria: State criteria used for exclusion of any data or subjects. Policies for statistical analysis should be included in the Information for Authors. Journals should have mechanisms to check for statistical accuracy.

1. Experimental uncertainty, statistics "There are three kinds of lies: lies, damned lies, and statistics." Attr. (by Mark Twain) Benjamin Disraeli, British Prime Minister Transparency is the key…....

1. Experimental uncertainty, statistics JBC Instructions to Authors: Authors must include information on uncertainty and reproducibility of data in figure legends. Authors must state numbers of independent samples (biological replicates) and replicate samples (technical replicates) analyzed and report how many times each experiment was repeated. Variation/precision should be reported by standard deviation (SD) (preferable), confidence intervals (CI) or standard error of the mean (SEM).

1. Experimental uncertainty, statistics Motulsky, J. Pharmacol. Exp. Ther. 351:200 Same data analyzed and plotted differently:

1. Experimental uncertainty, statistics Instructions to Authors: Animal studies State whether or not animals and/or samples analyzed were randomized, and indicate how. State whether studies were blinded or not. If so, indicate the method of blinding. State whether any data were excluded. If so, indicate the reason and/or criteria for exclusion.

1. Experimental uncertainty, statistics CC BY-NC 2.5 license.

2. Graphical presentation of data Weissgerber et al., PLOS Biology 13: e Mean ± SEM bar graphs can be misleading

2. Graphical presentation of data Weissgerber et al., PLOS Biology 13: e Mean ± SEM bar graphs can be misleading

2. Graphical presentation of data Instructions to authors: Scatter plots strongly recommended for small data sets; box and whisker plots for large data sets.

3. Western blots Instructions to authors: Define species of origin and source of all antibodies used, including catalog/lot numbers. Describe how novel antibodies were generated, including preparation/purification of epitope/antigen. Describe data supporting antibody specificity, including post-translational modifications or neoepitopes. If possible, demonstrate loss of immunoreactivity following genetic or other molecular modification to the antigen.

3. Western blots Instructions to authors: Immunoblots should be cropped in a way that retains information about antigen size, antibody specificity. Cropped images should ideally include positions of molecular weight markers above AND below the band(s) of interest. Avoid assembling figures of blots by splicing lanes from different sections of a gel. If blots must be spliced, borders must be clearly marked and explained in the figure legend.

3. Western blots Keep original data and unprocessed scans!

3. Western blots

Instructions (semi-quantitative analyses): Explain how data were obtained, whether signal intensity was linear with antigen loading, and how protein loading was normalized. Some detection methods (e.g., ECL) have a very limited linear range. Strongly prefer normalizing signal intensity to total protein loading (assessed by staining membranes for total protein). “House-keeping” proteins should not be used for normalization without evidence that manipulations do not affect expression. Phospho-specific antibody signals should be normalized to total levels of the target protein.

3. Western blots Instructions to authors: During the editorial review process, authors may be asked to provide high resolution images of original immunoblots, quantification details, and antibody validation data. Keep your original data and the unprocessed scans!

“The revision process provided us an opportunity to learn more about using total protein as a loading control, and we are glad to learn that the total protein staining of the Western blots is a superior and simpler method. We will use this method in our ongoing and future experiments/projects.” Author response in a rebuttal Supported by a large literature making the same point.

Implementation 1.Clearly define expectations and policies 2.Author education 3.Consistency in reviews (by EBMs!)