Fig 2: CBFB is a tumor suppressor and essential for maintaining RUNX1 protein levels. a IB showing expression of WT and CBFB mutants in CBFB_KO MCF10A cells. b Hematoxylin & eosin (H&E) staining of a representative xenograft tumor formed from subcutaneously injected CBFB_KO MCF10A cells. c IB showing the reduction of RUNX1 protein in CBFB_KO MCF10A cells. d IB showing RUNX1 deletion in MCF10A cells. e H&E staining of a representative tumor formed from RUNX1_KO MCF10A cells. f IB showing the subcellular localization of CBFB and RUNX1 in multiple breast cells. GAPDH, a marker for the cytoplasm (c); histone H3, a marker for the nucleus (N). g immunocytochemistry (ICC) showing the subcellular location of CBFB and RUNX1 in MCF10A cells. h IB showing the effect of RUNX1 deletion on the subcellular distribution of CBFB between the cytoplasm and nucleus. The numbers underneath the CBFB blot indicate the relative CBFB amounts quantified using ImageJ. i Co-immunoprecipitation (Co-IP) showing the interaction of RUNX1 with a N-terminal FLAG tag (F-CBFB) or a C-terminal tag (CBFB-F) in MCF10A cells. j IB showing the effect of overexpression of F-CBFB or CBFB-F on RUNX1 protein levels in CBFB_KO MCF10A cells

Fig 3: CBFB and hnRNPK regulate translation. a Flow chart showing the in vitro translation assay to test the effect of a translation element (RNA) and a protein. In vitro transcribed RNA was used in the in vitro translation assays using HeLa cells lysate. b In vitro translation assay using the 1-Step Human coupled IVT kit showing the effect of CBFB and hnRNPK binding sites (T14) on RUNX1 translation. c In vitro translation assays using the 1-Step Human coupled IVT showing effect of recombinant CBFB (rCBFB) and hnRNPK (rhnRNPK) on RUNX1 translation. d In vitro translation assay using WT and CBFB_KO HEK 293 T cells lysate. See “Methods” section for details of preparation of 293 T cell lysate. e In vitro translation assay. Un-transfected (control) and hnRNPK knockdown (KD) HEK 293T cells lysate replaced the HeLa cell lysate in the 1-Step Human coupled IVT kit with or without the supplement of rhnRNPK. f IB showing the effect of CBFB deletion on the proteins encoded by bound transcripts. g IB showing the effect of hnRNPK KD on the proteins encoded by bound transcripts

Fig 4: CBFB binds to RUNX1 mRNA via hnRNPK. a Silver staining of FLAG pulldown using CBFB KO MCF10A cells expressing empty vector and N-terminally FLAG tagged CBFB. b IB validation of CBFB and hnRNPK interaction. c IB showing the effect of hnRNPK knockdown on RUNX1 protein. d RNA immunoprecipitation (RIP) with CBFB antibody in WT and CBFB_KO MCF10A cells. Error bars are SEM, n = 3 (biological); two asterisks, p value < 0.01. (RIP of CBFB in WT vs. in CBFB KO cells, two-tailed t test). e RIP with hnRNPK antibody. Error bars are SEM, n = 3 (biological); two asterisks, p value < 0.01 (RIP of hnRNPK in WT vs. CBFB KO cells, two-tailed t test). f RNA pulldown assays (RPA) determining the hnRNPK-bound region within RUNX1 mRNA. F1-4, fragment 1 to 4 of 3′ UTR of RUNX1 mRNA. See Methods for details. Numbers indicate the nucleotide positions. g Re-analyses of two public eCLIP datasets (GSM2423241 and GSM2423242) of hnRNPK on the RUNX1 locus. Nucleotide sequences of two poly-C tracts within the binding site of hnRNPK were shown. F3, fragment 3 of 3′-UTR; T14, truncation 14 of 3′-UTR F3; Mu1, mutation 1; Mu2, mutation 2. h RPA using recombinant CBFB in the absence or presence of recombinant hnRPNK. i RPA using recombinant hnRNPK in the absence or presence of recombinant CBFB

Fig 5: 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