ROS production non-cell autonomously promotes pro-cancer processes in adjacent cells. (A) A diagram describing the mammalian cell model used to examine the impact of ROS production on transcription in adjacent cells. In this model, we co-cultured ROS-producing HEK293 cells expressing (DAAO expressing) with HEK293 cells expressing GFP in the presence of D-amino acids for 8 h. We then isolated the GFP+ cells via flow cytometry and purified mRNA for RNA-seq. All subsequent data is based on three independent biological replicates. All genes that were identified as differentially regulated display an FDR < 0.05). (B) A heat map depicting the expression ratio (ROS-coculture/control) for genes associated with the GO term innate immunity. (C) A heat map depicting the expression ratio (ROS-coculture/control) for genes known to be directly regulated by the RBPL/Notch signaling pathway. (D) A table of Gene ontology terms enriched in genes downregulated(top) or upregulated (bottom) by ROS production in adjacent cells. (E) A heat map depicting the expression ratio (ROS-coculture/control) for genes associated with the GO term extracellular matrix. (red indicates known association with cancer growth and metastasis). (F) A heat map depicting the expression ratio (ROS-coculture/control) for genes associated with the GO term transcription factor (red indicates known association with cancer growth and metastasis). (G) A heat map depicting the expression ratio (ROS-coculture/control) for genes associated with the GO term small cell lung cancer. (H) A model summarizing the transcriptional changes we observed in cells co-cultured adjacent to ROS-producing cells. Student’s t test was used for all pairwise comparisons, and one-way ANOVA was used for all experiments containing >2 sample groups. Error bars represent the standard deviation.