Nature Genetics: doi: /ng Supplementary Figure 1
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1 Supplementary Figure 1 MSI2 interactors are associated with the riboproteome and are functionally relevant. (a) Coomassie blue staining of FLAG-MSI2 immunoprecipitated complexes. (b) GO analysis of MSI2-interacting protei. (c) Proportion of total read counts sequenced per pool. cntl, pool. (d) Scatterplot depicting the concordance of shrna abundance (log 2 normalized read counts) between each one of the replicates and its respective group (median log 2 normalized read counts). (e) Diagrams showing the number of hairpi represented with a minimum arbitrary threshold (normalized counts >100). (f) Waterfall plot depicting normalized depletion levels for all shrnas in the spleen (SP). Control shrnas and hairpi targeting selected candidate genes are highlighted. (g) Table showing seven candidate genes. (h) Validation of efficient knockdown of target genes in mouse MLL-AF9 leukemia cells. Cells were selected under puromycin treatment for 48 h before qrt PCR. Actb serves as a housekeeping gene. All data represent the mea + s.e.m. of at least three independent experiments. P < 5, P < 1, P < 01, two-tailed t test.
2 Supplementary Figure 2 SYNCRIP is required for survival of leukemia cells in vitro and in vivo. (a) Efficient knockdown of SYNCRIP in mouse MLL-AF9 leukemia cells. MLL-AF9 cells were traduced with lentiviruses expressing shrna (directed agait luciferase) or shrnas directed agait Syncrip (shrna 1 and shrna 2). Cells were selected under puromycin for 24 h before immunoblotting. Cells were collected and assayed 3 d after viral traduction. Actin serves as a loading. (b) shrna depletion of SYNCRIP promoted myeloid differentiation of leukemia cells. Myeloid differentiation status was assayed by FACS analysis of F-480 and CD115 expression. A representative FACS plot is shown of and SYNCRIP-KD leukemia cells. (c) Quantitative summary of FACS analysis of F-480 and CD115 expression in and SYNCRIP-KD leukemia cells 3 and 4 d after traduction. (d) Quantitative summary of FACS analysis of the percentage of c-kit high cells in and SYNCRIP-KD leukemia cells 4 d after traduction. (e) Increased apoptosis was not observed in SYNCRIP-KD leukemia cells at 3 and 4 d after traduction. Quantitative summary of Annexin-V assessed by flow cytometry. (f) Annexin-V assessed by flow cytometry 5 d after traduction of and SYNCRIP-KD leukemia cells. Representative of FACS analysis in Figure 2e. (g) qpcr showing efficient knockdown of Syncrip in mouse AML1-ETO9a-driven leukemia cells. Cells were selected under puromycin for 48 h before qrt PCR. Actb serves as a housekeeping gene. (h) Cells from g were plated into methylcellulose and scored for the number of colonies (average of three independent experiments). (i) Assessment of disease burden, including spleen weight and liver weight, for the recipient mice in Figure 2f. (j) Immunoblots showing SYNCRIP expression in the bone marrow of mice that succumbed to disease in i and Figure 2f. (Mice injected with Syncrip-KD1 with reduced disease burden (group 1) maintained a better Syncrip knockdown level than mice that manifested similar disease to the group (group 2).) (k) qpcr showing efficient knockdown of Syncrip assayed by primers designed for specific grna targeting regio (grna-specific primers) in RN2 cells traduced with CRISPR Cas9-containing Tetinducible grnas specific for Syncrip (grna1 grna3) in comparison to, Cas9-EV. (l) Quantitative summary of FACS analysis of
3 the percentage of Gr-1 + and Mac-1 + cells in the Cas9-EV- and Syncrip-gRNA-traduced leukemia cells in Figure 2i. (m) Quantitative summary of FACS analysis of the percentage of c-kit high cells in the Cas9-EV- and Syncrip-gRNA-traduced leukemia cells Figure 2i. (n) Immunoblot analysis of RN2 cells overexpressing different isoforms of SYNCRIP corresponding to protei of 527, 562 and 623 aa. The SYNCRIP 562-aa isoform was the predominantly expressed isoform in leukemia cells. Actin serves as a loading. (o) SYNCRIP overexpression in RN2 cells promotes colony formation (average of three independent experiments). (p) qpcr showing mouse Syncrip expression as assayed by grna-specific primers and human SYNCRIP expression for Figure 2k. (q) Immunoblot analysis of the RN2 cells overexpressing SYNCRIP or carrying the (empty vector) traduced with CRISPR Cas9-containing Tet-inducible grna1 and grna3 cotructs or Cas9-EV in Figure 2j. All data represent the mea + s.e.m. of at least three independent experiments. P < 5, P < 1, P < 01, two-tailed t test.
4 Supplementary Figure 3 Syncrip-deleted hematopoietic cells retain colony-forming and in vivo engraftment capacity. (a) Representative PCR analysis for genotyping of Syncrip deletion in fetal liver cells. (b) Quantitative summary of FACS analysis of hematopoietic stem and progenitor cells in the WT and Syncrip-CR-KO fetal livers in Figure 3c (WT n = 7, CR-KO n = 7). (c) Quantitative summary of the number of all colony types formed by WT and Syncrip-CR-KO fetal liver cells (average of six biological samples). BFU-E, burst-forming unit erythroid; M, macrophage; G, granulocyte; GM, granulocyte, macrophage; GEMM, granulocyte, erythroid, macrophage, megakaryocyte. (d) Assessment of disease burden, including spleen weight and liver weight, for the recipient mice in Figure 3j. P < 5, two-tailed t test.
5 Supplementary Figure 4 SYNCRIP is expressed in human AML, and SYNCRIP depletion results in apoptosis and increased myeloid differentiation in AML cell lines. (a) The graph shows the log 2 expression of SYNCRIP from tracript profiling of bone marrow cells from healthy donors and patients with various types of hematological malignancies, including ALLs, B-ALLs, CLL, MDS, CML and subtypes of AML. Hypodiploid B-ALL, n = 40; ALL with t(1;19), n = 36; ALL with t(12;21), n = 58; B-ALL with t(8;14), n = 13; c-/pre-b-all without t(9;22), n = 237; c-/pre-b-all with t(9;22), n = 122; pro-b-all with t(11q23)/mll, n = 70; CLL, n = 448; T-ALL, n = 174; MDS, n = 206; CML, n = 76; complex AML, n = 48; AML with inv(16), n = 28; AML MLL, n = 38; AML with a normal karyotype, n = 351; AML with t(15;17), n = 37; AML with t(8;21), n = 40; healthy donors, n = 73. P < 1, P < 01, P < 001, Student s t test. (Hemaexplorer data for SYNCRIP probe _s_at from the U133 and U133 Plus 2.0 arrays.) (b) qpcr showing SYNCRIP mrna levels in multiple human AML cell lines and normal cord blood derived CD34 + (CB-CD34 + ) cells. ACTB served as a housekeeping gene. Relative mrna level was normalized to the SYNCRIP mrna level in CB-CD34 + cells. (c f) Immunoblots showing efficient SYNCRIP knockdown in the indicated human AML cell lines. (g) Quantitative summary of CD14 high and CD13 high cells and CD14 and CD13 MFI in MOLM13, NB4 and NOMO- 1 cells traduced with shrna or shrnas agait SYNCRIP (shrna 1 and shrna 2) 4 d after traduction. (h) Representative FACS plots of the cells in Figure 4h. All data represent the mea + s.e.m. of at least three independent experiments. P < 5, P < 1, P < 01, two-tailed t test.
6 Supplementary Figure 5 Elevated expression of SYNCRIP correlates with expression profiles of the HOXA9 and MEIS1 target genes IKZF2 and MYC in patients with AML. (a) Plots showing the distribution of mean normalized counts of KD1 correlating with normalized mean counts of KD2 from the RNA sequencing data in Figure 5a. (b) Normalized read counts of Syncrip, Hoxa9, Myc, Ikzf2 and Meis1 from the RNA sequencing data in Figure 5a. (c f) log 2 mrna levels of the indicated genes in patients with high versus low SYNCRIP mrna expression in the data sets for patients with AML reported in Figure 4a (high SYNCRIP was defined as individuals with a value greater than the average + 1 s.d. while low SYNCRIP was defined as individuals with a value greater than the average 1 s.d.). (c) MSI2 mrna. (d) Target genes upregulated by HOXA9 MEIS1. (e) Target genes downregulated by HOXA9 MEIS1. (f) IZKF2 mrna. (g) MYC mrna. P < 5, P < 1, P < 01, two-tailed t test.
7 NOMARLIZED TOTAL RNA a b c MOLM13 cells - puromycin 4 days post traduciton d dsred cells - 3 days post traduction KD-1 KD-2 e dsred cells - 4 days post traduction KD-1 KD-2 f dsred cells - 4 days post traduction co ntro l KD -1 MSI2 KD -2 MSI2 Syncrip Hoxa9 c-myc Ikzf2 Syncrip Hoxa9 c-myc Ikzf2 SYNCRIP HOXA9 c-myc ACTIN g dsred cells - 4 days post traduction MSI2-KD1 MSI2-KD2 h RN2 cells - 24h after induction Cas9-EV grna1 grna2 i 2.0 MOLM13 cells- 3 days post traduction shrna#1 shrna#2 j MOLM13 cells- 4 days post traduction 3 2 shrna#1 shrna#2 1 Msi2 Syncrip Hoxa9 c-myc Ikzf2 Hoxa9 c-myc Ikzf2 SYNCRIP HOXA9 c-myc IKZF2 0 SYNCRIP c-myc HOXA9 IKZF2 k KD-1 KD-2 Cas9 TOTAL RNA grna-1 grna-2 shrna#1 shrna#2 dsred cells RN2 cells MOLM13 cells l m KD-2 KD-1 Syncrip Syn3 Syn1 Cas9 Syncrip Hoxa Hoxa c-myc c-myc Ikzf Ikzf Supplementary Figure 6 SYNCRIP post-tracriptionally s HOXA9 expression. (a) MOLM13 human myeloid leukemia cells carrying the MLL-AF9 tralocation overexpressing MSI2 were immunoprecipitated for endogenous MSI2 and SYNCRIP. (b) Immunoblots showing protein expression of HOXA9, MYC and IKZF2 upon SYNCRIP knockdown in dsred MLL-AF9 cells 3 and 4 d after traduction. Actin serves as a loading. (c) Immunoblots showing protein expression of HOXA9 and MYC upon SYNCRIP knockdown in MOLM13 cells after 2 d of puromycin selection (4 d after traduction). (d,e) qpcr measuring the mrna expression of Syncrip, Hoxa9, Myc and Ikzf2 in dsred tertiary MLL-AF9 leukemia cells 3 and 4 d after traduction. (f) Immunoblots showing protein expression of HOXA9, MYC and SYNCRIP upon MSI2 knockdown in dsred MLL-AF9 cells at 4 d after traduction. (g) qpcr measuring mrna expression of Syncrip, Hoxa9, Myc and Ikzf2 upon MSI2 knockdown in dsred MLL-AF9 cells at 4 d after traduction. (h) qpcr measuring mrna expression of Syncrip, Hoxa9, Myc and Ikzf2 in RN2 cells 24 h after induction. (i) qpcr measuring mrna expression of SYNCRIP, HOXA9, MYC and IKZF2 in human MOLM13 leukemia cells 3 d after traduction. (j) qpcr measuring mrna expression of SYNCRIP, HOXA9, MYC and IKZF2 in human MOLM13 leukemia cells 4 d after traduction. (k) Normalized total RNA levels in dsred MLL-AF9, RN2 and MOLM13 cells upon SYNCRIP depletion. (l) mrna stability of Syncrip, Hoxa9, Myc and Ikzf2 in dsred cells traduced with and SYNCRIP shrnas 4 d after traduction. (m) mrna stability of Syncrip, Hoxa9, Myc and Ikzf2 in RN2 cells expressing Cas9-EV or grna1 and grna3 targeting Syncrip after 24 h of Dox induction. Actin served as a loading. Actb served as a housekeeping gene. All data represent the mea + s.e.m. of at least three independent experiments. P < 5, P < 1, P < 01, two-tailed t test.
8 Supplementary Figure 7 HOXA9 but not MYC overexpression partially rescues the colony-forming ability of SYNCRIP-depleted cells. (a) qpcr measuring mrna expression showing that MSI2 overexpression increased the mrna levels of Hoxa9, Myc and Ikzf2. (b) Immunoblots showing efficient depletion of SYNCRIP and protein expression of HOXA9 for the cells in a. (c) Colony formation was rescued in dsred SYNCRIP-KD leukemia cells overexpressing full-length HOXA9. (d) Immunoblots showing efficient depletion of SYNCRIP and protein expression of HOXA9 for the cells in c. (e) Colony formation was not rescued in dsred SYNCRIP-KD leukemia cells overexpressing MYC. (f) Immunoblots showing efficient depletion of SYNCRIP and protein expression of MYC for the cells in e. (g) Cell growth was not rescued in MOLM13 SYNCRIP-KD cells overexpressing MYC. (h) Immunoblots showing efficient depletion of SYNCRIP and protein expression of MYC for the cells in g. Actin served as a loading. Actb served as a housekeeping gene. All data represent the mea + s.e.m. of at least three independent replicates. P < 5, P < 1, P < 01, P < 001, two-tailed t test.
Control shrna#9 shrna#12. shrna#12 CD14-PE CD14-PE
a Control shrna#9 shrna#12 c Control shrna#9 shrna#12 e Control shrna#9 shrna#12 h 14 12 CFU-E BFU-E GEMM GM b Colony number 7 6 5 4 3 2 1 6 pm A pa pc CFU-E BFU-E GEMM GM pu pgm A p pg B d f CD11b-APC
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