Fig. 4: CNOT3 controls translation of c-MYC.

A Experimental scheme of omic profiling by RNA-sequencing and proteomic analysis by tandem mass tag (TMT) mass spectrometry. MOLM-13 cells were transduced with lentiviruses expressing either a scramble (control) shRNA or CNOT3-targeting shRNAs (KD33 and KD-37). Cells were selected for puromycin resistance and assayed 2 days post-transduction. n = 3 independent experiments. B, C Gene set enrichment analysis (GSEA) of B transcriptomic profiling and C proteomic analysis of CNOT3 depleted cells vs. control as described in A. D Immunoblot confirming efficient knockdown of CNOT3 and reduction of c-MYC upon CNOT3 depletion in MOLM13 cells. ACTIN serves as loading control. E Quantitative qRT-PCR assessment of mature c-MYC transcript and primary (unprocessed) c-MYC transcript in MOLM13 cells. F Quantitative qRT-PCR measure of c-MYC abundance over time after treatment of cells with actinomycin D to inhibit transcription. ACTIN serves as housing keeping gene control. G Polysome profiling of CNOT3 depleted cells vs. control. Graphs showing optical density profiles (ODA₂₅₄) of RNA across polysome gradients. Mono di-some and polysome fractions are shown. H Quantitative qRT-PCR measure of c-MYC abundance in input and relative abundance in polysome vs. monosome fractions. All n = 3 independent experiments, two-tailed Student’s t test, ns not significant. All graphs show data as mean ± s.e.m, n = 3 independent experiments, p values calculated by two-tailed student’s t test. Source data are provided as Source Data files for figures (D–F, H).