Differential regulation of transcriptional and metabolic profiles in DN-POLG cells in the course of inducible mtDNA depletion.

<p>RNA-seq was performed from DN-POLG cells at days 0, 3, 6 and 9 of doxycycline supplementation; <i>N</i> = 3 biological replicates per time point. Transcriptional profiling was based on DEGs identified using log2-transformed fold-changes in RPKM versus the mean at day 0 (Log2FC) in two-way ANOVA tests (gene × time) at an adjusted Benjamini-Hochberg <i>p</i> ≤ 0.05. (A) Unsupervised clustering analysis of log₂-fold expression changes versus average of day 0 for DEGs observed in DN-POLG cells by RNA-seq; increased (red) or decreased (green) expression. (B) Number of DEGs identified in DN-POLG cells at days 3, 6 and 9 of doxycycline supplementation compared to day 0 (circle plots, top). (C) Schematic representation of the main transcriptional responses associated to DEGs in DN-POLG cells at days 3, 6 and 9 of dox-inducible mtDNA depletion in DN-POLG cells, per IPA. (D) Schematic representation of interconnected amino acid and methionine metabolic pathways associated to differentially enriched metabolites at days 3, 6, and 9 of dox-inducible mtDNA depletion in DN-POLG cells (<i>N</i> = 4 per time point), per IPA. (E–H) Box plots of relative content versus day 0 (IQR-outlier format) depict distribution of individual replicates in DN-POLG cells within statistical groups for (E) IMP, Xan, XMP, Cho, Met, Leu, Put, 4-HPPA, (F) Ser, (G) f-Met, and (H) Cys; partial gray backgrounds highlight metabolite relative contents at day 3 in (E). Underlying data are reported in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005707#pbio.2005707.s007" target="_blank">S1 Data</a> for (A) and (B); <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005707#pbio.2005707.s008" target="_blank">S2 Data</a> for (C) and (D); and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005707#pbio.2005707.s009" target="_blank">S3 Data</a> for (E–H). 4-HPPA, 4-hydroxyphenylpyruvate; Cho, choline; Cys, cysteine; DEGS, differentially enriched genes; DN-POLG, dominant-negative DNA polymerase gamma transgene; ETC, electron transport chain; f-Met, N-formylmethionine; GABA, γ-aminobutyric acid; IMP, inosine 5'-monophosphate; IPA, Ingenuity Pathway Analysis; Leu, leucine; Met, methionine; mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation; Put, putrescine; RNA-seq, RNA sequencing; ROS, reactive oxygen species; RPKM, reads per kilobase per million; Ser, serine; TCA, tricarboxylic acid; Xan, xanthine; XMP, xanthosine 5'-monophosphate.