## Replication Study: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology <br> **Abstract:** As part of the [Reproducibility Project: Cancer Biology][1], we published a Registered Report ([Khan et al., 2015][2]), that described how we intended to replicate selected experiments from the paper "A coding-independent function of gene and pseudogene mRNAs regulates tumour biology" ([Poliseno et al., 2010][3]). Here we report the results. We found *PTEN* depletion in the prostate cancer cell line DU145 did not detectably impact expression of the corresponding pseudogene *PTENP1*. Similarly, depletion of *PTENP1* did not impact *PTEN* mRNA levels. The original study reported *PTEN* or *PTENP1* depletion statistically reduced the corresponding pseudogene or gene (Figure 2G; [Poliseno et al., 2010][3]). *PTEN* and/or *PTENP1* depletion in DU145 cells decreased PTEN protein expression, which was similar to the original study (Figure 2H; [Poliseno et al., 2010][3]). Further, depletion of *PTEN* and/or *PTENP1* increased DU145 proliferation compared to non-targeting siRNA, which was in the same direction as the original study (Figure 2F; [Poliseno et al., 2010][3]), but not statistically significant. We found *PTEN* 3'UTR overexpression in DU145 cells did not impact *PTENP1* expression, while the original study reported *PTEN* 3'UTR increased *PTENP1* levels (Figure 4A; [Poliseno et al., 2010][3]). Overexpression of *PTEN* 3'UTR also statistically decreased DU145 proliferation compared to controls, which was similar to the findings reported in the original study (Figure 4A; [Poliseno et al., 2010][3]). Differences between the original study and this replication attempt, such as level of knockdown efficiency and cellular confluence, are factors that might have influenced the results. Finally, where possible, we report meta-analyses for each result. ---------- ### Contents **Reports:** Read the [Replication Study][10] or view the [earlier versions][11]. **Note**: In order to successfully run and knit the Replication Study Manuscript R Markdown file, you must install a series of necessary R packages. You can review the necessary packages included in the checkpoint manifest in the markdown file [here][12] or run and knit the following r markdown file and they will be downloaded automatically. To reproduce the Replication Study manuscript text run this in [R Studio][13] (note: downloads [R markdown file][12] directly from osf.io: library(httr) library(rmarkdown) GET("https://osf.io/v3cag/?action=download",write_disk("Replication_Study_1.Rmd", overwrite = T)) render("Replication_Study_1.Rmd", output_format = "word_document") <br> Also, explore the Registered Report and materials related to the Registered Report [here][2]. **Data and Material Availability:** - Plasmids generated during this study are deposited and available at [Addgene][14]: pCMV-PTEN-3'UTR (plasmid# [97204][15]) - All other associated data, protocols, analysis scripts, and other digital materials are available within this OSF project. **Experiments replicated**: Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. <br> - [Cell growth assay following siRNA transfection][17] - Replication of Figure 2F of Poliseno et al., 2010 <br> - [Quantitative PCR following siRNA transfection][18] <br> - Replication of Figure 2G of Poliseno et al., 2010 - [Western blot assay following siRNA transfection][19] <br> - Replication of Figure 2H of Poliseno et al., 2010 - [Quantitative PCR following *PTEN* 3′ UTR transfection][20] <br> - Replication of Figure 4A left panel of Poliseno et al., 2010 - [Cell growth assay following *PTEN* 3′ UTR transfection][21] <br> - Replication of Figure 4A right panel of Poliseno et al., 2010 **Meta-analysis**: As a measure of evaluating reproducibility a meta-analysis of each effect was performed. The forest plots, analyses, and data are available [here][22]. <br> Questions about the project can be directed to [contact+rpcb@cos.io.][23] [1]: https://osf.io/e81xl/wiki/home/ [2]: https://elifesciences.org/articles/08245 [3]: https://doi.org/10.1038/nature09144 [10]: https://elifesciences.org/articles/51019 [11]: https://osf.io/hbvdn/ [12]: https://osf.io/v3cag/ [13]: https://www.rstudio.com [14]: https://www.addgene.org [15]: https://www.addgene.org/97204/ [17]: https://osf.io/kjmxj/wiki/home/ [18]: https://osf.io/4uard/wiki/home/ [19]: https://osf.io/re87y/wiki/home/ [20]: https://osf.io/rkuxh/wiki/home/ [21]: https://osf.io/jgp6n/wiki/home/ [22]: https://osf.io/9yh6p/wiki/home/ [23]: mailto:contact+rpcb@cos.io