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. 2021 Jun 23;11:13158. doi: 10.1038/s41598-021-92448-w

Figure 3.

Figure 3

Depletion of USO1 leads to decreased cell growth, cell cycle arrest and increased apoptosis. (A) Schematic representation of the pLKO5.sgRNA.EFS.tRFP lentiviral vector. Abbreviations, hU6, human U6 promoter; sgRNA, short guide RNA; sg scaf, sgRNA scaffold; tRFP, turbo red fluorescent protein. (B) Sample FACS plots of SEM cells transduced sequentially with Cas9 vector and sgRNA containing vector. Left, non-transduced SEM cells; middle, transduced with pLentiCas9-GFP; right, cells transduced with both pLenti-Cas9-GFP and pLKO5 vector containing USO1-targeting sgRNA. (C) Western blot for USO1 in SEM cells following CRISPR/Cas9-mediated disruption of the USO1 gene using three different sgRNAs (sg1-3) and NT, non-targeting sgRNAs. (D) RT-qPCR measurement of USO1 in control (NT) and USO1 (sg2 & sg3) SEM cells (t test; **P < 0.01; ****P < 0.0001) (E) MTS assay to study the cell growth of USO1-depleted cells (sg2 & sg3), measured as Absorbance at 490 nM (t test; **P < 0.01; ***P < 0.001). (F, G) Cell cycle analysis using propidium iodide (PI) staining of control cells and USO1-depleted cells (F) and quantitation of cells from cell cycle analysis (Two-way Annova with Bonferroni correction; *P < 0.05; ****P < 0.0001). (H, I) FACS plots of Annexin V positivity in control versus USO1-depleted cells (H), Quantitation of cells with Annexin V positivity (t test; **P < 0.01) (I).