Fig. 4. CDK12 overexpression induces aberrant metabolic phenotypes in normal mammary MCF10A cells.

a KEGG enriched pathways of differentially upregulated genes in RNAseq (n = 2) of CDK12-OE vs. EV MCF10A cells. Metabolic pathways are in red. b Hierarchical clustering heatmap of the entire set of metabolites differentially expressed in the steady-state profile of CDK12-OE vs. EV MCF10A cells by t-test analysis (see abbreviations in Supplementary Table 1). Three independent samples performed in triplicate for each condition were analyzed by LC-MS. The color code scale indicates the normalized metabolite abundance (ranging from −2 up to 2). The clusters containing EV and CDK12-OE MCF10A cells are highlighted in green and red, respectively. c Pathway impact analysis of steady-state metabolite profiling as in (b). Color intensity (white-to-red) and size of each circle reflects increasing statistical significance, based on the P-value [-log(P)] from the pathway enrichment analysis (y-axis) and the pathway impact value derived from the pathway topology analysis (x-axis), respectively. Gluconeo., gluconeogenesis; TCA, tricarboxylic acid; hypotau., hypotaurine; GSH, Glutatione. d Schematic of the metabolic pathways upregulated in CDK12-OE vs. EV MCF10A cells showing derivation and contribution of carbon atoms in intermediate metabolites traced with ¹³C-Glucose. Key genes involved in these pathways are also indicated. Red circles, ¹³C-labeled carbon atoms. Solid arrows represent direct metabolic reactions, and dashed arrows represent multiple reactions and indirect connections between two metabolites. Outputs of the different metabolic routes are colored in red. SSP, serine synthesis pathway. Met. Salv., methionine salvage. e Isotopolog abundance of serine (Ser), glycine (Gly), 5,10 Me-THF, 5,10-MTHF in CDK12-OE vs. EV MCF10A cells grown in medium containing ¹³C-Glucose (17.5 mM). Data are the mean ± SD (n = 3), represented as percentage isotopomer labeling relative to control condition. ***P < 0.001 vs. EV, two-sided unpaired t-test. m + n: mass of the isotopomer + n, where n represents the number of heavy carbons (¹³C). Source data are provided as Source Data file.