Fig. 3

Validation of top pseudogene hits in MCF7 cells. A A bar graph shows the log₂FC of sgRNA abundance difference between day 21 and day 0 for the top-ranked (by log₂FC) pseudogene hits in MCF7 cells that showed a significant upregulation in breast cancer compared with normal breast tissues based on TCGA data. B qRT-PCR analysis of the RNA level of MGAT4EP, DDX12P, PRELID1P1, and TUBBP5 in MCF7-dCas9 cells transduced with negative control non-targeting sgRNA (sg-NT) or gene-specific sgRNA. GAPDH was used as an internal control. The growth of MCF7-dCas9 cells transduced with sg-NT or gene-specific sgRNA for C MGAT4EP, D DDX12P, E PRELID1P1, and F TUBBP5 was monitored (OD450 absorbance for WST-8 formazen) every 24 h with CCK-8 assay for 96 h. G The representative pictures of clonogenic growth and H the bar graph quantifying the colonies formed by MCF7-dCas9 cells transduced with sg-NT or gene-specific sgRNAs for MGAT4EP, DDX12P, PRELID1P1, and TUBBP5, after cells were cultured for 2 weeks. All data are shown as mean ± standard deviation (SD), n = 3. The Student’s t test was used to assess the statistical significance of difference in mean between two experimental groups (*p < 0.05; **p < 0.01; ns: not significant, p ≥ 0.05)