Fig. 6. eIF4A3 knockdown promotes 80S monosome–mediated translation of stress-related genes and induces p53-mediated cell cycle arrest.

(A) OPP incorporation followed by high-content microscopy for the quantification of translation rates in U2OS treated with sieIF4A3 or CHX as control. A total of 1000 to 2000 cells were analyzed per experiment (data are shown as means ± SD; n = 3 biological replicates; *P < 0.05, ****P < 0.001). (B) Polysome profiling of U2OS cells following eIF4A3 knockdown and separation of the cell lysates in sucrose gradients. High-lightened regions depict fractions used in downstream analyses (magenta, 80S monosome; cyan, polysomes consisted of more than three ribosomes). (C) Starburst plot comparing DE genes in RNA and protein level following sieIF4A3 treatment of U2OS cells (R = 0.56, P < 0.001). (D) Graphic illustration of the experimental design for polysome profile RNA-seq and proteomic analysis. (E) UpSet plot among the DE genes affected by sieIF4A3 in the 80S monosome or polysome translation level. Blue, the common targets; magenta, the genes associated with 80S monosomes; cyan, the ones bound to the polysomes. (F) Starburst plot comparing DE genes in the translation (80S monosome) and protein level following sieIF4A3 treatment of U2OS cells (R = 0.45, P < 0.001, axis of quadrants I to III). (G) Representative 5-ethynyl-2′-deoxyuridine (EdU)–4′,6-diamidino-2-phenylindole (DAPI) IF intensity scatter plots following Click-IT EdU immunostaining of U2OS cells treated with sieIF4A3 ± ActD^L (>1000 cells were analyzed per condition). Green points represent cells in S phase. (H) Cell cycle staging in U2OS following treatment with sieIF4A3 ± ActD^L. The analysis was based on EdU incorporation and cyclin A1 immunostaining. A total of 1000 to 2000 cells were analyzed per experiment (data are shown as means ± SD; n = 3 biological replicates; **P < 0.01, ***P = 0.01, and ****P < 0.001; nonsignificant values are not shown).