Fig 2: Effect of depletion of eIF3 subunits on cell and tumor growth Parental HCT116 and the five different conditional eIF3‐mAID cell lines were treated with the vehicle DMSO or with IAA for the periods indicated. Metabolic activity (i.e., NAD(P)H) as a proxy of cell proliferation was determined by MTT assay. Graphs represent means ± SD, n = 5–6. Asterisks denote: *P < 0.005, **P < 0.00005, ***P < 0.000005 (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli).1 × 103 cells of the indicated cell lines were plated and grown in DMSO or IAA for 2 weeks. Colony numbers were counted and are shown in a bar graph relative to the DMSO‐treated control. Note that very low numbers of colonies were obtained for IAA‐treated eIF3a‐mAID, eIF3e‐mAID and eIF3f‐mAID – hence no bars are visible. Bars represent means ± SD, n = 3. Numbers indicate P‐values (unpaired Student's t‐test).The indicated cell lines were subjected to serum withdrawal (0% FBS) for 16 h. DMSO or IAA was added and cells were stimulated to re‐enter the cell cycle by refeeding with media containing 10% FBS. Cells were fixed after the indicated time points, and the cellular DNA content was measured by flow cytometry. Data are representative of 2 (HCT116, eIF3a‐mAID, eIF3b‐mAID) or 3 (eIF3e‐mAID, eIF3f‐mAID, eIF3k‐mAID) experiments.1 × 106 eIF3‐mAID cells were injected into nude mice. When tumors reached a diameter of ~ 4 mm, mice were treated with vehicle or 500 mg/kg IAA, and tumor growth was measured for 2 weeks. Graphs represent means ± SD, n = 5–7. Numbers indicate P‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli).Expression of the indicated eIF3 proteins in 97 colon adenocarcinomas versus 100 normal colonic mucosae and 110 clear cell renal cell carcinomas versus 84 normal kidney samples. Log2 spectral count data from CPTAC (Chen et al, 2019) via UALCAN portal (Chandrashekar et al, 2022). Data information: The median (50th percentile), 25% percentile (lower box), 75th percentile (upper box), 5th percentile (lower whisker), and 95th percentile (upper whisker) are indicated. n = biologically independent tumor samples. Source data are available online for this figure.

Fig 3: Effect of depletion of eIF3 subunits on mRNA translation The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunits. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Signals were normalized to the reference signal of actin and plotted relative to the vehicle DMSO. Bars represent means ± SD, n = 3. Numbers indicate P‐values (unpaired Student's t‐test).The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (M) and polysomal (P) peaks were quantified, and P/M ratios relative to the vehicle DMSO were plotted. Bars represent means ± SD, n = 3. Numbers indicate P‐values (unpaired Student's t‐test).Scatter plots of changes in polysomal mRNA versus total mRNA upon depletion of the indicated eIF3 subunits.Enrichment of Gene Ontology terms in the sets of polysome‐associated mRNAs decreased in the polysomal fractions of eIF3f‐ or eIF3e‐depleted cells or increased in eIF3k‐depleted cells.Distribution histograms of translational efficiencies (TEs) of the mRNA sets shown in (C). The total number of mRNAs and the median TE of each dataset are indicated. The number of up and down changes with FDR ≤ 0.2 as well as the median of all changed items are indicated in red, blue, and black bold print.Distribution histograms of the protein synthesis ratios determined in eIF3 subunit depleted versus replete cells. The total number of measured proteins and the median synthesis change of each dataset are indicated. The number of up and down changes with FDR ≤ 0.1 as well as the median of all changed items are indicated in red and blueprint. Data information: n = number of biological replicates. Source data are available online for this figure.