Fig. S1. Nature Medicine: doi: /nm HoxA9 expression levels BM MOZ-TIF2 AML BM. Sca-1-H. c-kit-h CSF1R-H CD16/32-H. Mac1-H.

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1 A CSF1RH CSF1RH CSF1RH GFPH Sca1H ckith CSF1RH CSF1RH CSF1RH Mac1H CD16/32H Gr1H Cell counts CSF1R high G/G1 SG2/M Hoechst blue Cell counts CSF1R low G/G1 SG2/M Hoechst blue C HoxA9 expression levels ormal H L CSF1R M TIF2 AML M Fig. S1 ature Medicine: doi:1.138/nm.2122

2 A lue TIF2 AML M GFP gated Verapamil non SP SP lue Hoechst Red Hoechst Red CSF1R SP CSF1R high 92.6 GFP gated CSF1R onsp CSF1R high Mac Mac C leukemia free survival (%) non SP D leukemia free survival (%) SP Time (weeks) Time (weeks) Fig. S2 ature Medicine: doi:1.138/nm.2122

3 A CSF1Rhigh CSF1Rlow control AP counts counts counts GFPH GFPH GFPH 1 weeks 3 months Fig. S3 ature Medicine: doi:1.138/nm.2122

4 A myeloid.fcs FSCH, SSCH subset 1 4 myeloid.fcs FSCH, SSCH subset 1 4 myeloid.fcs FSCH, SSCH subset CSF1R MSCFRH Mac1H Gr1 Gr1H GFPH GFPH GFP GFPH GFP C D leukemia free survival (%) 1 8 AP control Time (days) leukemia free survival (%) CSF1R/ CSF1R/ Time (days) Fig. S4 ature Medicine: doi:1.138/nm.2122

5 STAT5 pstat5 ormal M AML M csf1r / / / ormal M AML M / / / pstat5 STAT5 5 Fig. S5 ature Medicine: doi:1.138/nm.2122

6 A Interaction with WT 124 H P HAT acidic S M H P HAT acidic PQ T acidic S M d H P S M T acidic T acid 1664 H P 1585 H P 1513 H P 1443 H P 1392 H P 1311 H P P S M S S S Localization /C [kda] WT d IP C [kda] IP D [kda] IP E [kda] IP F [kda] G [kda] IP H [kda] IP 25 Fig. S6 ature Medicine: doi:1.138/nm.2122

7 A WT DE Q PEST ETS ΔDE Q PEST ETS ΔQ DE PEST ETS ΔDEQ PEST ETS ΔPEST DE Q ETS ΔEts1 DE Q PEST ΔEts2 DE Q PEST iing to / / F W:HA IP:FLAG W:HA IP:FLAG W:FLAG WT ΔDE ΔQ ΔDEQ ΔPEST ΔEts1 ΔEts2 HA C F WT ΔDE ΔQ ΔPEST ΔEts2 D F WT ΔDE ΔQ ΔPEST ΔEts2 HA 1513 W:HA HA W:HA IP:FLAG W:HA IP:FLAG W:HA E Relative luc activity (fold) TIF2 CP * ** ** TIF2 CP TIF2 CP TIF2 CP * ** ** TIF2 CP TIF2 CP WT ΔDE ΔQ ΔDEQ ΔPEST ΔEts1 Fig. S7 ature Medicine: doi:1.138/nm.2122

8 [kda] (1%) GST GST GST S(1664) 5 Fig. S8 ature Medicine: doi:1.138/nm.2122

9 A Transactivation Transactivation Interaction with WT H P HAT acidic S M PQ H P HAT acidic d144 dh15 dphd d dhat d66511 TIF2 P HAT acidic S M P HAT acidic S M H HAT acidic S M H P T acidic S M H P acidic S M H P HA acidic S M H P HAT a d H P T a TIF2 TIF2 d66511 H P HA a TIF2 Localization CP Interaction with C/H romo C/H H P HAT acidic HAT Q H P HAT acidic HAT H P HAT acidic HAT H P HAT acidic dphd dhat d66511 H HAT acidic H P HA acidic HAT HAT H P acidic HAT Q HAT Q Q R R R R R R Relative luc activity (fold) ** WT d144 dh15 dphd ** d ** dhat d66511 C Relative luc activity (fold) ** ** TIF2 WT ** TIF2 d * TIF2 d66511 D Relative luc activity (fold) ** ** ** ** * CP WT CP CP CP CP dphd CP dhat CP d66511 Fig. S9 ature Medicine: doi:1.138/nm.2122

10 qrtpcr Iuction (fold) Csf1r mra 2 4HT (h) ER ER TIF2 Fig. S1 ature Medicine: doi:1.138/nm.2122

11 A Relative occupancy ChIP M/TIF2AML control α α control α α Myf5 Csf1r Relative occupancy Csf1r 4HT IgG α α IgG α α PUER PUER/TIF2 Fig. S11 ature Medicine: doi:1.138/nm.2122

12 A / fcs MCSFRPEH, LineageioAviAPCCy7H s / fcs MCSFRPEH, LineageioAviAPCCy7H s 6 6 counts 4 counts MCSFRPEH CSF1R MCSFRPEH CSF1R qrtpcr 16 Csf1r mra Gapdh mra Relative expression level Relative expression level / / / / Fig. S12 ature Medicine: doi:1.138/nm.2122

13 Supplemental Figure Leges Fig. S1. CSF1R high LICs were ckit a Mac1 low. (A) The Ms from TIF2 iuced AML mice were analyzed by FACS for expression of GFP, CSF1R, Mac1, ckit, Sac1, CD16/32, a Gr1 (A). The Ms were also stained with CSF1R a Hoechst. CSF1R high a CSF1R low/ cells were analyzed for cell cycle distribution (). (C) CSF1R high a CSF1R low/ cells were sorted a analyzed for HoxA9 expression by qrtpcr. Fig. S2. Side population (SP) cells are CSF1R high in TIF2iuced AML mice. (A) The M cells of TIF2iuced AML mice were stained with Hoechst in the presence or absence of verapamil, a SP cells were analyzed by FACS. () The M cells from AML mice were stained with anti CSF1RPE, anti Mac1PECy7, a Hoechst GFP SP cells (right panel) a GFP nonsp cells were analyzed for expression of CSF1R a Mac1. (C, D) Iicated numbers of flowsorted nonsp cells (C) a SP cells (D) were transplanted into sublethally irradiated mice, a leukemiafree survival was investigated. Fig. S3. (A) The M cells from transgenic (Csf1rEGFPGFR/FKP1A/TFRSF6) mice were infected with MSCVTIF2iresGFP a were transplanted into lethally irradiated C57L/6 mice to iuce AML. CSF1R high a CSF1R low/ cells were sorted a analyzed for morphology by staining with MayGiemsa. () The M cells (1 1 5 ) of primary AML mice were transplanted into sublethally irradiated C57L/6 mice. Administration of AP2187 or solvent (control) to secoary AML mice was begun by IV injection three weeks after transplantation. Expression of GFP was analyzed by FACS 1 week or 3 months after start of administration. Fig. S4. MYC iuced AML mice. The M cells from MYC iuced AML mice were analyzed by FACS for expression of GFP, CSF1R, Mac1, a Gr1 (A) a for morphology by staining with MayGiemsa (). (C) The M cells from transgenic (Csf1rEGFPGFR/ ature Medicine: doi:1.138/nm.2122

14 FKP1A/ TFRSF6) mice were infected with MSCVTIF2iresGFP, a transplanted into lethally irradiated C57L/6 mice to iuce AML. Intravenous administration of AP2187 or solvent (control) to secoary AML mice was begun three weeks after transplantation. Leukemiafree survivals of the untreated (n = 6) a AP2187treated (n = 6) secoary transplanted mice were investigated (P =.7348). (D) Fetal liver cells of E16.5 CSF1R/ a CSF1R/ mice embryo littermates were infected with MYCiresGFP, a transplanted into irradiated mice. Leukemiafree survivals of the mice were analyzed. n = 5, P =.534 Fig. S5. Phosphorylation of STAT5. Fetal liver cells of E16.5 CSF1R / a CSF1R / mice embryo littermates were infected with MYCiresGFP, a transplanted into irradiated mice. The M cells from normal mice or CSF1R / a CSF1R / AML mice were analyzed for expression of STAT5 a phosphorylated STAT5 by immunoblot with antistat5 (Santa Cruz, sc181) a antiphosphospecific STAT5 (Cell Signaling, #8359) antibodies. Fig. S6. Interaction of mutants with. (A) Structure for deletion mutants of. The results for interaction with or AML a the nuclear () or nuclear/cytoplasmic (/C) localization are iicated., not determined (H) 293T cells were transfected with wildtype a mutants of HAtagged together with FLAGtagged or FLAGtagged. Immunoprecipitates with antiflag antibody (IP:FLAG) or cell lysates () were subjected to immunoblotting with antiha or antiflag antibodies. The mutant containing only the Cterminal region (151724) interacts with but not. It is possible that the mutant does not accumulate in the nucleus, a thus would not be able to form complexes with. Fig. S7. Functional domains of (A) Structure for deletion mutants of. (D) Interaction of mutants with. 293T cells were transfected with HAtagged fulllength (), 1513 (C), or (D) together with wildtype a mutants of FLAGtagged. Immunoprecipitates with antiflag antibody (IP:FLAG) or cell lysates () were subjected to immunoblotting with antiha or antiflag antibodies. (E) SaOS2 ature Medicine: doi:1.138/nm.2122

15 cells were transfected with the CSF1R luciferase construct a the effectors iicated. Luciferase activity was analyzed 24 h after transfection. Error bars represent SD (n = 3). * P <.1 a ** P <.5. Fig. S8. Interaction of with a. The HiIIIClaI fragment correspoing to the terminal region (1664) of was cloned into psp64polya vector. [35S](1664) protein was produced using the TT Coupled Rabbit Reticulocyte Lysate System (Promega) with [35S]methionine, a was tested for interaction with GST, GST, a GST, which were produced in E. coli L21 containing pgex6p, pgex6p, a pgex6p, respectively. The input lane represents 1% of the material used for biing to the GST fusion protein. Fig. S9. The domain of a fusions required for CSF1R transactivation. (A) Structure for deletion mutants of, TIF2 a CP. The a interacting domain a the results for transacrivation (, ) or repression (R) of or mediated transcription a nuclear () localization are iicated., not determined (D) SaOS2 cells were transfected with the CSF1R luciferase construct a together with deletion mutants of, TIF2, a CP. Luciferase activity was analyzed 24 h after transfection. Error bars represent SD (n = 3). * P <.1 a ** P <.5. Fig. S1. depeent iuction of CSF1R mras by TIF2. PUER cells infected with MSCVGFP, or MSCVTIF2iresGFP retroviruses were exposed to 1 nm 4hydroxytamoxifen (4HT) for 24 or 48 h a analyzed by quantitative RTPCR for expression of CSF1R mra. Fig. S11. Chromatin immunoprecipitation (ChIP) of a. The M cells from TIF2iuced AML mice (A), or PUER cells infected with MSCVGFP (PUER) or MSCVTIF2iresGFP retroviruses (PUER/TIF2) () were tested by ChIP analysis using antiterminal region of, anti antibodies, a control rabbit IgG. PUER a PUER/TIF2 cells were incubated with () or without () 4HT for 48 h. Semiquantitative ature Medicine: doi:1.138/nm.2122

16 realtime PCR was performed on the precipitated DAs using the following primers: CSF1R (5 GCA TCG CTG TCC TGC AGT AG3, 5 AAA TCT GCA CTC TTA TGC CAG AGA3 ), Myf5 (5 GGA GAT CCG TGC GTT AAG AAT CC3, 5 CGG TAG CAA GAC ATT AAA GTT CCG TA3 ) Fig. S12. Expression of CSF1R in / mice. (A) Fetal liver cells of E14.5 / a / mouse embryo littermates were analyzed by FACS for expression of CSF1R. () Reverse transcription a quantitative PCR were performed as described previously using TaqMan probes for Csf1r (Mm432689_m1) a Gapdh (Mm _g1) purchased from Applied iosystems. Expression levels normalized to those of Gapdh were calculated using a staard curve a the relative quantification method described in AI User ulletin #2. ature Medicine: doi:1.138/nm.2122