After publication of this article [1], questions were raised about some of the reported results:

• In Figure 2A there appears to be a discontinuity in background after lane 1 of the β-actin blot, when brightness and contrast are adjusted. The discontinuity is also visible in the original underlying image for this blot (S1 File), which matches the image in the published figure. The original Pitx and TUJ1 blot images supporting Figure 2A are in S2 and S3 Files, respectively. Lanes 1–6 and 8–13 in each of these image files show data from the same blot and exposure. Quantitative data supporting the graph in Figure 2A are in S4 File, and the results of quantification using TUJ1 control data are in S5 File.
• In Fig 4C, there is a vertical discontinuity between lanes 1, 2 in the pPitx3 panel. The authors noted that an extra control lane from the original image was spliced out when preparing the figure and apologized for not having indicated the splicing clearly in the published figure and its legend. An updated Fig 4 is provided here in which this has been addressed. The original image data underlying the pPitx3 results in Fig 4C are in S6 File. Fig 4C shows a representative result from experiments using 0.5 μg antibody per ChIP, and the overall results for ChIP-Real time PCR experiments using 2 μg antibody per ChIP are summarized quantitatively in Fig 4D. Quantitative data supporting pPitx3 results in Fig 4D are in S7 File, and replication data for this experiment are in S8 File. Raw quantification data for the BDNF experiment represented in Fig 4D are no longer available. The following issues arose in our editorial review of the data files provided in support of Fig 4C and 4D:
  • Among the replicate data (S6 File), the data shown in Fig 4C show the weakest band for IgG and the strongest band for Nurr. As such, the data observed across experimental replicates provides weaker support for the difference between IgG and Nurr1 compared to images in Fig 4C. The quantitative data in Fig 4D show the cumulative results from multiple experimental replicates.
  • During the editorial assessment of the quantitative data, questions were raised about the different results shown in S7 File for experiments done using 0.5, 1, 2, or 4 μg of input RNA: unlike the 0.5 and 2 μg conditions (2 μg results are reported in the article), the 1 and 4 μg data do not appear to support enrichment over background. The authors commented that they did not repeat the 4 μg experiments for which they estimated there were saturating amounts of antibody that may have impacted background. The authors also confirmed that the 1 μg antibody immunoprecipitation was less efficient compared to the 0.5 and 2 μg experiments, and they noted that they included only the 0.5 and 2 μg conditions in replicate experiments.

The corresponding author stated that original data underlying all other results reported in the article are available upon request.

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Fig 4. Nurr1 directly regulates Pitx3 expression by binding to its promoter.

(A) ChIP-PCR analysis performed in MN9D transfected with 3×FLAG-Nurr1 and immunoprecipitated with anti-FLAG antibody shows a significant enrichment of Pitx3, Bdnf and Vmat2 promoter regions. No enrichment was observed when an unrelated region of the Vmat2 promoter was used [19]. The inserts show representative PCR amplified fragments after ChIP. (B) ChIP-Real time PCR in MN9D transfected with 3×FLAG-Nurr1 and immunoprecipitated with anti-FLAG antibody. The diagram shows the fold enrichment over background (dotted line) for Pitx3, Bdnf and Vmat2 promoter regions. (C) ChIP-PCR validation performed in E14.5 midbrain and immunoprecipitated with Nurr1 antibody shows a significant enrichment of the Pitx3 and Bdnf promoter regions. A representative PCR amplified fragment is shown into the insert. The vertical black line in the pPitx3 panel indicates an image splice junction: the panel shows data obtained using the same original gel and exposure, additional lanes were removed when preparing the figure. The original gel image is in S6 File. (D) The diagram shows the ChIP-Real time PCR quantitation of Bdnf and Pitx3 promoter region in E14.5 midbrain and immunoprecipitated with Nurr1 antibody. Results are expressed as mean ± SE of at least three independent experiments. Asterisks (*) represent p≤0.01 when compared to control (ANOVA, Scheffè F-test).

https://doi.org/10.1371/journal.pone.0233918.g001