Supplemental Figure 1
A. ** 5 MIG-R1/NGFR 4 MIG-uHC/NGFR * 3
2
1 Relative Recovery
0 24 48 72 Time (hours)
B. 24hrs 48hrs 72hrs 30 80 60
60 20 40
40 Cell Numbers 10 20 20
0 0 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K
FSC
C. shLuc shMyb
c-Myb
β-Actin
1 0.2
Supplemental Figure 1. Efficacy of the uHC transduction and Irf4/8-/- large pre-B cell models. Pro-B cells from Mybff Rag2-/- mice were co-transduced with MIG-R1 or MIG-17.2.25 (uHC) and NGFR or NGFR-Cre and cultured with 10ng/ml IL-7. (A) Total numbers of co-transduced GFP+ NGFR+ cells was analyzed 24, 48, and 72 h post-transduction by flow cytometry. Relative recovery was determined by normalization to the total number of co-transduced cells at the 24 h time point. Retrovirus transductions were done in triplicate. *p < 0.05, **p < 0.005. (B) Forward scatter (FSC) histograms of co-transduced GFP+ NGFR+ cells from (A). The solid line represents MIG-uHC/NGFR transduced cells (pre-B) and the dotted line represents MIG-R1/NGFR transduced cells (pro-B). Data is representative of 2 independent experiments. (C) Irf4/8-/- large pre-B cells were transduced with shLuc- GFP or shMyb-GFP and cultured with 10ng/ml IL-7. GFP+ cells were electronically sorted at 72 h post- transduction and knockdown efficiency of c-Myb protein was analyzed by Western blot. β-Actin was used as a loading control. Data is representative of 3 independent experiments. Supplemental Figure 2
A. B. KEGG_CELL_CYCLE (Log2 Fold Change) KEGG_GLYCOLYSIS_GLUCONEOGENESIS (Log2 Fold Change) -1.0 -0.5 0.0 0.5 1.0 1.5 -1 0 1 2
CDKN1C ALDH1A3 CDKN1B ZBTB17 LDHB CDKN2D ALDOC HDAC1 CCND2 ALDH3A2 E2F2 ALDH1B1 CDK7 CCNE1 PFKM MCM4 HK2 MCM7 ALDH2 CCNB2 CCNA2 PGK1 MCM2 PGAM1 MCM3 CDK1 PDHB MCM5 DLAT HDAC2 PCNA GAPDH CHEK1 DLD CCNB1 PDHA1 CDK4 CCNH AKR1A1 E2F5 ADH5 CCND3 MCM6 ALDH7A1 CCNE2 HK1 CDKN1A MYC ALDH9A1
C. D.
KEGG_OXIDATIVE_PHOSPHORYLATION (Log2 Fold Change) GO_LYMPHOCYTE_DIFFERENTIATION (Log2 Fold Change) -1 0 1 2 -2 -1 0 1 2
ATP4A IRF4 TCIRG1 IL2RA ATP6AP1 BCL6 ATP6V0B CD2 COX8A COX6B1 SYK COX10 IKZF1 COX6A1 STAT5B SDHA EP300 UQCRH PIK3CD ATP6V1G1 RAG1 COX4I1 UQCRC1 STAT3 SDHC SOX4 SDHB CD19 CYC1 FNIP1 UQCRB LCK UQCR10 CD79A UQCRC2 ATP6V1H JAK3 UQCRFS1 IKZF3 UQCRQ TSC1 COX7B CD79B PPA1 LIG4 SDHD TCF3 COX6C COX5B LEF1 UQCR11 IL7R COX5A ZAP70 COX7C KIT Supplemental Figure 2. Gene set enrichment of c-Myb-dependent differentially expressed genes. c-Myb-dependent gene expression changes in large pre-B cells were identified by RNA-seq of Irf4/8-/- large pre-B cells after shMyb transduction as described in Fig. 4. Differential expression of genes identified from gene set analysis described in Fig. 4B in the Cell_Cycle (A), Glycolysis_Gluconeogenesis (B), and Oxidative_Phosphorylation (C) KEGG pathway gene sets and the Lymphocyte_Differentiation (D) GO gene set is shown. Genes upregulated upon c-Myb knockdown are shown in black and genes downregulated upon c-Myb knockdown are shown in red. Supplemental Figure 3
5 N.S. pMSCV *** 4 N.S. IkWT *** Ik159A 3
N.S. N.S. 2 N.S. *** N.S. *** 1 Relative Fold Change 0 c-Myc Foxo1 Txnip Hk1 Slc2a1
Supplemental Figure 3. Effects of wild type Ikaros and Ik159A on gene expression in pre-B cells. Microarray differential expression analysis of B3 pre-B cells transduced with pMSCV, IkWT, or Ik159A was performed as described in Ferreiros-Vidal et al, Blood (2013). Log2 fold change values comparing pMSCV empty vector transduced cells to IkWT or Ik159A transduced cells were converted to fold changes with the empty vector control set as 1. Significance was determined by Benjamini-Hochberg adjusted p-value comparing the pMSCV empty vector to fold changes induced by IkWt or Ik159A transduction. Supplemental Table I
Quantitative Real Time PCR Primers Gene Forward Primer Reverse Primer Ccnd3 ATGCTGGAGGTGTGTGAGGA CCACAGCCTGGTCCGTATAG Foxo1 AAGGATAAGGGCGACAGCAA TGGATTGAGCATCCACCAAG Hk1 GCCACGCCTCGGTGCCATCTT GGTCTTGTGGAACCGCCGGG Hprt TGCCGAGGATTTGGAAAAAGTG CACAGAGGGCCACAATGTGATG Ikzf1 (Ikaros) GGAGGCACAAGTCTGTTGAT CATTTCACAGGCACGCCCATTCT Ikzf3 (Aiolos) GCCGAGATGGGAAGTGAGAG CCGGGATTGTAGTTGGCATC Myc CGAAACTCTGGTGCATAAACTG GAACCGTTCTCCTTAGCTCTCA Rag1 GGGGAGTGGGGTTGAAAGTA TCCTCCAATCCTGCCTCCTA Slc2a1 AGCCCTGCTACAGTGTAT AGGTCTCGGGTCACATC (Glut1) Txnip CGAGTCAAAGCCGTCAGGAT TTCATAGCGCAAGTAGTCCAAAGT