Fig 2: CBFB interacts with and facilitates mRNA binding of eIF4B. a FLAG IP followed by IB showing the interaction of translation initiation factors with CBFB. 10 µg/ml RNase A was added in the co-IP lysate. b Endogenous co-IP followed by IB showing interaction of CBFB, hnRNPK, and eIF4B. 10 µg/ml RNase A was added in the co-IP lysate. c In vitro translation assays showing the effect of rCBFB, rhnRNPK, and reIF4B on RUNX1 translation in a cap-dependent or -independent manner. d RIP followed by real-time PCR to show the effect of CBFB deletion on the binding of eIF4B on CBFB-bound transcripts. Error bars are SEM, n = 3 (biological); n.s., p value > 0.05 (RIP of eIF4B in WT vs. CBFB KO cells). e A model showing the role of CBFB in translation regulation through hnRNPK and eIF4B

Fig 3: CBFB regulates translation initiation. a Protein absorbance of each fraction of ribosome fractionation of WT and CBFB_KO MCF10A cells. b Real-time PCR measuring the abundance of RUNX1 mRNA in each fraction. Shown are the percentages of total RUNX1 mRNA in each fraction. Error bars are SEM, n = 3 (biological repeats). c Percentage of total RUNX1 mRNA in the monosome and pooled polyribosomal fractions. Numbers on top of brackets are the ratios of RUNX1 mRNA amount in pooled poly- to monoribosomal fractions. Error bars are SEM, n = 3 (biological), two asterisks, p value < 0.01 (CBFB KO vs. WT comparisons in the free and polysome fractions, two-tailed t test). d Real-time PCR measuring the percentage of total GAPDH mRNA in each fraction. Error bars are SEM, n = 3 (biological repeats). e Percentage of total RUNX1 mRNA in the mono- and pooled polyribosome fractions. Numbers are the ratios of GAPDH mRNA amount in pooled poly- to monoribosomal fractions. Error bars are SEM, n = 3 (biological); n.s., p value > 0.05 (CBFB KO vs. WT comparisons in the free and polysome fractions, two-tailed t test). f Real-time PCR measuring percentage of each transcript in the free, mono- and polyribosomal fractions in WT and CBFB_KO MCF10A cells. Error bars are SEM, n = 3 (biological); n.s., p value > 0.05. g Bar chart showing the percentage of group 1 and group2 in CBFB/hnRNPK-bound mRNAs. Group 1, ratio of polyribosomal vs. free fraction decreased more than half in CBFB_KO compared to WT cells; Group 2, the rest of mRNAs. The expected percentage is based on the global mRNAs. Chi-square test was used to calculate the p value

Fig 4: Genome-wide binding of CBFB and hnRNPK to mRNAs. RIPseq of FLAG-CBFB (a) and hnRNPK (b) in MCF10A cells. Shown are mean adjusted FPKM + 1 (log2). Red dots show the transcripts that are enriched in FLAG-CBFB RIP more than 4-fold. The rest transcripts are shown as blue dots. c A Venn diagram showing transcripts bound by both CBFB and hnRNPK. d Validation of binding of CBFB and hnRNPK to 16 selected transcripts using RIP followed by real-time PCR. Error bars are SEM, n = 3 (biological). e The top-ranked gapped motif, which was represented in 86% of the hnRNPK binding sites occurred in hnRNPK-bound transcripts

Fig 5: The transcriptional function of CBFB is critical for breast tumor suppression. a A Venn diagram showing genes (identified by RNAseq) co-regulated by CBFB and RUNX1. b Kaplan-Meier curve showing 10-gene signature upregulated by CBFB and RUNX1 predicts disease-free survival of breast cancer patients. c IB showing the upregulation of Notch intracellular domain (NICD) of NOTCH3 in CBFB_KO and RUNX1_KO cells. d ChIPseq of RUNX1 showing the binding of RUNX1 on the NOTCH3 locus. e Anchorage independent growth assays of double knockout (DKO) of CBFB (C) and NOTCH3 (N) or DKO of RUNX1(R) and NOTCH3(N). n = 3 (biological); two asterisks, p value < 0.01 (single KO vs. double KO comparisons, two-tailed t test). f Representative IHC images showing the protein levels of CBFB, RUNX1, and NOTCH3 in human breast tumors and normal tissues. g IB showing the effect of exogenous expression of CBFB on RUNX1 and NOTCH3 expression in MCF7 and BT474 cells. h Weight and (i) volume of orthotopically transplanted tumors from MCF7 cells that were transduced with an empty vector (EV) or a lentiviral vector expressing CBFB (CBFB). Tumor incidence is shown on the top