Fig. 3
Ectopic BORIS expression transforms NIH3T3 cells and deregulates gene transcription. a Representative images of soft agar colonies. b Quantification of the number of soft agar colonies. Two-tailed Student’s t test (***—p < 0.0001). c Western blot analysis of BORIS expression in nuclear extracts isolated from four single-cell clones of NIH3T3 cells, recovered from a soft agar. K562—positive control. Parental (WT) and EV-expressing NIH3T3 cells were used as a negative control for BORIS expression. RAD21 Abs were used as a loading control. d RT-qPCR results displaying the relative expression levels of testis-specific Gal3St1 in NIH3T3 + EV, including EV-total culture (#1), and single-cell clones with EV (#2, #3), compared to NIH3T3 + BORIS clones recovered from soft agar (#1, 2, 3, 4). Two-tailed Student’s t test ( *—p < 0.005). Error bars indicate mean ± SD (n = 3). e Heatmap depicting BORIS occupancy in three BORIS-expressing clones (#2,3,4) compared to EV. f Quantification of the number of upregulated (red) and downregulated (blue) RefSeqGenes, lncRNAs, and TEs in three BORIS-expressing clones (#2,3,4), compared to EV, with log2 fold change > 1.3 and p-value (padj) < 0.05. g–k NIH3T3 cells were stably transfected with either the doxycycline-inducible empty vector (pBIGi-EV) or BORIS (pBIGi-BORIS). The cells were cultured under different conditions: in the absence of doxycycline (No Dox), induced with doxycycline (Dox) for a specified number of hours, and doxycycline removal (Wash Off) for an indicated number of hours. g Western blot confirms BORIS activation by dox. h Heatmap of BORIS occupancy in doxycycline-treated cells compared to EV and cells without doxycycline treatment. i Quantification of upregulated (red) and downregulated (blue) RefSeqGenes in doxycycline-induced EV or BORIS cells compared to EV (no dox) or EV (dox 12 h), respectively, with log2 > 1.3, p-value < 0.001. k The heatmap depicts the expression profile of 1377 genes (RNA-seq data, 2 replicates for each condition), which were significantly deregulated in BORIS-expressing cells treated with dox for 24 h, comparing the expression in EV versus BORIS-induced cells. l GSEA of RNA-seq data for BORIS-expressing cells isolated from soft agar, compared to EV