Figure 5.

USP9X ablation or inhibition impairs the ribosomal stalling response. (A) HEK293 Flp-In T-Rex GFP-P2A-(K^AAA)₂₁-P2A-RFP WT cells were transfected with a plasmid containing Cas9 and gRNAs targeting USP9X or ZNF598. P, pool of USP9X guides; 1/2/4/7, individual USP9X guides; Z, ZNF598 guide. Lysates were analyzed 168 h after transfection and selection in puromycin by immunoblotting with the indicated antibodies. Panel is representative of two independent experiments. (B) Schematic of the fluorescent ribosomal stalling reporter expressed in this cell line. If stalling is not efficiently resolved, read-through occurs, and the FLAG-SR and RFP are expressed. (C) FACS analysis of the RFP:GFP ratio in (K^AAA)₂₁ WT or ZNF598 KO cells following transfection with px459-pSpCas9(BB)-2A-Puro_v2–containing gRNA as indicated. Cells were gated for live singlets, then for GFP-positive cells. Graphs depict data from >7,000 cells. Insets indicate the USP9X protein levels normalized to WT untransfected cells. (D) FACS analysis of the RFP:GFP ratio in WT cells following inhibition of USP9X with 10 µM FT709 for 72 h. Graph depicts data from >8,000 cells and is representative of three independent experiments. (E) (K^AAA)₂₁ WT cells were treated with indicated concentrations of FT709 for 48 h and analyzed by immunoblotting with selected antibodies (representative of three independent experiments). (F) quantitation of Makorin mRNA levels for cells treated as in E. Error bars in E and F indicate the standard deviation (n = 3 independent experiments); two-tailed Student’s t test; **, P < 0.01; ***, P < 0.001. Arrows in A and E indicate two isoforms of MKRN1, and the upper one is quantified. TPS, total protein stain.