Loss of RIOK2 leads to decreased protein synthesis followed by apoptosis. (A) Quantification of FACS-based EdU labeling and calculation of cell cycle distribution of MA9 cells 6 days after transduction with lentiviruses expressing the indicated sgRNAs. n = 3 for each sgRNA. Error bars represent standard error of the mean. (B) Two representative FACS plots of EdU-labeled MA9 cells expressing either NC-1 or Riok2 sgRNAs. (C) OP-puro labeling followed by FACS analysis of cells transduced with lentiviruses expressing Riok2, Rps19, or NC-1 sgRNAs. The translation inhibitor cycloheximide was used as a positive control. Analysis was performed on FACS-sorted cells 4 days after sgRNA transduction. The Student t test was used to assess statistical significance. **P < .01; ****P < .0001. n = 2-3 biological replicates. Error bars represent SD. (D) Overview of the RNA-Seq experiments using MA9 Riok2^fl/fl R-Cre^ER treated with either OHT (n = 3 biological replicates) or EtOH (n = 3 biological replicates) for 72 hours. (E) Volcano plot showing upregulated and downregulated transcripts after loss of RIOK2. Significantly changed transcripts (P < .05; log₂ FC > ±1) are shown in red. (F) Gene set enrichment analysis of apoptosis hallmark gene signatures comparing OHT vs EtOH treated Riok2^fl/fl R-Cre^ER MA9 cells. (G) Gene Ontology term analysis on significantly downregulated genes upon 72 of OHT treatments of Riok2^fl/fl R-Cre^ER MA9 cells (log₂FC <−1; P < .05).