Fig. 5: E2F6 and ZNF274 overexpression each induce an aged transcriptome in hGPCs.

scRNA-sequencing of E2F6 and ZNF274 overexpression (OE) with a GFP lentivirus used as control, 7 days following doxycycline (Dox) treatment. A UMAPS of isolated GPC populations showing PDGFRA expression in addition to E2F6 and ZNF274 for demonstration of overexpression (EGFP CTR: 921 GPCs; E2F6 OE: 1085 GPCs, ZNF274 OE—906 GPCs). B Venn diagram of both differentially expressed genesets (FDR < 0.05, Log₂FC > 0.25). C, D Upset plots indicating repressed genes shared between fetal vs adult hGPCs and E2F6 I or ZNF274 (D) OE cells, and their respective targets, as determined via rCisTarget from these scRNA-seq OE experiments and the fetal vs adult bulk RNA-seq experiments. E Scoring via AUCell of the rCisTarget determined regulons of either E2F6 or ZNF274. Black lines indicate median expression. F, G Heatmaps indicating Log₂ fold changes and significances of adult vs. fetal GPCs (F) and E2F6 or ZNF274 vs EGFP CTR (G). H Significant direct targets of E2F6 or ZNF274 OE. I qPCR validation of select direct targets (indicated in blue in F; n = 4 biologically independent samples). Central lines indicate the median, boxes show the interquartile range, and the whiskers indicate 1.5 times the interquartile range. Outliers are plotted beyond the whiskers. J Dot plot of curated IPA terms (FDR < 0.05). K KI67 staining of EGFP⁺ cells (Dox Ctr is of DAPI⁺, n = 3 biologically independent samples). L. β-Galactosidase⁺ staining of DAPI⁺ cells (n = 3 biologically independent samples). Statistics for I, K, and L were calculated via estimated marginal means tests of linear models, following regression of cell batch effect. FDR adjusted p-values: *<0.05, **<0.01, ***<0.001, ****<0.0001. Source data are provided as a Source Data file; exact p values are listed there. Error bars ± SEM.