Figure 3.
Some RPS15 mutants replace the endogenous protein and show altered proliferation and drug tolerance. (A) Schematic representation of RPS15 locus targeted by the designed single guide RNA (sgRNA), encompassing the second intron and the third exon of RPS15 gene and the corresponding locus in the pCDH-EGFP-RPS15 vector. The common sequence is highlighted in blue. (B) HEK293T cells stably expressing the different RPS15 constructs were infected with the lentiCRISPRv2 vector containing an sgRNA against endogenous RPS15 or no target (empty vector, Ø). Selected subclones were subjected to western blot analysis with antibodies against GFP (for GFP and GFP-RPS15 detection) and RPS15 and β-actin as a loading control. (C) Bar graph showing the percentage of clones with successful ablation of endogenous RPS15 in all of the different mutants, as well as in the wild-type and EGFP constructs. (D) Relative proliferation values of GFP-RPS15–expressing (wild-type and mutants) HEK293T cells transduced with the lentiCRISPRv2-empty (Ø) vector. (E) Relative proliferation values of GFP-RPS15–expressing (wild-type and mutants) HEK293T cells transduced with the lentiCRISPRv2-sgRNA against the endogenous RPS15 locus. Error bars indicate SEM. (F-H) Representative viability curves of selected clones of HEK293T cells transduced with the lentiCRISPRv2-sgRNA against endogenous RPS15 locus and treated with the indicated concentrations of ibrutinib (F), sorafenib (G), and fludarabine (H) for 72 h. (I) Summary table including the 50% inhibitory concentration (IC50 ± SEM) of the different RPS15 constructs using the aforementioned drugs. *P < .033, **P < .002, ***P < .001, two-way ANOVA.

Some RPS15 mutants replace the endogenous protein and show altered proliferation and drug tolerance. (A) Schematic representation of RPS15 locus targeted by the designed single guide RNA (sgRNA), encompassing the second intron and the third exon of RPS15 gene and the corresponding locus in the pCDH-EGFP-RPS15 vector. The common sequence is highlighted in blue. (B) HEK293T cells stably expressing the different RPS15 constructs were infected with the lentiCRISPRv2 vector containing an sgRNA against endogenous RPS15 or no target (empty vector, Ø). Selected subclones were subjected to western blot analysis with antibodies against GFP (for GFP and GFP-RPS15 detection) and RPS15 and β-actin as a loading control. (C) Bar graph showing the percentage of clones with successful ablation of endogenous RPS15 in all of the different mutants, as well as in the wild-type and EGFP constructs. (D) Relative proliferation values of GFP-RPS15–expressing (wild-type and mutants) HEK293T cells transduced with the lentiCRISPRv2-empty (Ø) vector. (E) Relative proliferation values of GFP-RPS15–expressing (wild-type and mutants) HEK293T cells transduced with the lentiCRISPRv2-sgRNA against the endogenous RPS15 locus. Error bars indicate SEM. (F-H) Representative viability curves of selected clones of HEK293T cells transduced with the lentiCRISPRv2-sgRNA against endogenous RPS15 locus and treated with the indicated concentrations of ibrutinib (F), sorafenib (G), and fludarabine (H) for 72 h. (I) Summary table including the 50% inhibitory concentration (IC50 ± SEM) of the different RPS15 constructs using the aforementioned drugs. *P < .033, **P < .002, ***P < .001, two-way ANOVA.

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