ice-cold 70% ethanol with gentle vortexing, incubated at -20 C for 4 hours, and washed with PBS.
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1 Cell cycle analysis For cell cycle analysis, single cell suspensions of E12.5 fetal liver cells were suspended in 4 ml ice-cold 7% ethanol with gentle vortexing, incubated at -2 C for 4 hours, and washed with PBS. Cells were then incubated with.2 mg/ml RNase (Invitrogen) / PBS at 37 C for 6 minutes. Propidium iodide (PI) was added at a final concentration of 5 µg/ml. Flow cytometry was carried out on a Becton Dickinson FACS LSR with CELLQuest (Becton Dickinson) software. Data were analyzed using FlowJo (Tree Star Inc., Ashland, OR) analysis software. Preparation of Native erythroblastic islands and Reconstituted erythroblastic islands Native erythroblastic islands were isolated from fetal livers using a previously described protocol 1. E13.5 fetal livers were dissected and dispersed by enzymatic digestion with collagenase S-1 (Nitta Gelatin, Osaka, Japan) and DNase I (Sigma-Aldrich, St. Louis, MO). Digested fetal liver cells (2 1 6 cells in 2 µl medium) were allowed to attach to glass coverslips for 2 minutes at 37 C in 5% CO 2. Attached cells were then flooded with medium and incubated for 4 hours. Adherent macrophages with attached erythroid clusters were obtained by dipping coverslips in RPMI to remove nonadherent cells and were then processed for immunofluorescence analysis ( Native erythroblastic island). To strip erythroblasts from macrophages, cells were incubated in PBS lacking Ca 2+ and Mg 2+ for 1 minutes. The remaining adherent fetal liver cells were allowed to re-spread in RPMI containing 1% FBS for 2 hours before receiving fresh erythroblasts (1 1 6 erythroblasts in 5 µl medium from c-maf +/+ or c-maf -/- E14.5 fetal liver). Finally, the reconstituted clusters were dipped to remove the unbound cells before being processed for immunofluorescence
2 ( Reconstituted erythroblastic island). Immunofluorescence staining was performed with rat anti-mouse F4/8 antibody (Serotec, Oxford, United Kingdom), visualized by Alexa 488-conjugated chicken anti-rat IgG (Molecular Probes, Eugene OR) and PE-conjugated anti-mouse TER-119 antibody (BioLegend, San Diego, CA). The number of erythroblasts attached to macrophages was determined by counting the total number of cells present in each island visualized using a Leica fluorescence microscope (Leica Microsystems, Tokyo, Japan). Four to six embryos were analyzed per combination group. For each combination, at least 2 macrophages per embryo were analyzed. Microarray analysis of fetal liver macrophages Fetal liver cells from E15.5 c-maf +/+ and c-maf -/- embryos were incubated with APC-conjugated anti-mac-1 antibody (ebioscience) and PE-conjugated anti-f4/8 antibody (Serotec). Mac-1 + F4/8 + cells were sorted by a FACSAria cell sorter (Becton Dickinson) as fetal liver macrophages. Total RNA was extracted from fetal liver macrophages using a QIAGEN RNeasy Mini Kit according to the manufacturer s instructions. The microarray procedure was performed as described in the Affymetrix GeneChip Expression Analysis Manual (Affymetrix). Biotin-labeled crna was synthesized from 4 to 6 µg RNA and hybridized to a GeneChip Mouse Genome Array (Affymetrix). After washing, arrays were stained with streptavidin-phycoethrin and analyzed on a scanner. Each array was analyzed using the GeneSpring (Agilent Technologies, Palo Alto, CA) program to calculate the average density for each probe set (Affymetrix). Data normalization and data analyses were performed using GeneSpring. The data have been deposited in the Gene Expression Omnibus database under the accession number GSE2335.
3 Table S1. Primer sequences used for PCR Primers Used for PCR Gene VCAM-1 forward VCAM-1 reverse EMP forward EMP reverse Rb forward Rb reverse IL-6 forward IL-6 reverse IL-1β forward IL-1β reverse IL-12b forward IL-12b reverse TNF-α forward TNF-α reverse Primer sequence 5 - GGA TGA ACA CTC TTA CCT GTG C -3' 5 - GGT CAA AGG GAT ACA CAT TAG GG -3' 5 - CCA CCA GAC ACA CAT ATC TC -3' 5 - CTG CAC ACA GGG CAG TCA GG -3' 5'- GTG ACA GAG TGC TCA AAA GA -3' 5'- ATC CTT GCT CTC ATT CAT TC -3' 5'- CAC AGA GGA TAC CAC TCC CAA CA -3' 5'- TCC ACG ATT TCC CAG AGA ACA -3' 5'- CAA CCA ACA AGT GAT ATT CTC CAT G -3' 5'- GAT CCA CAC TCT CCA GCT GCA -3' 5'- AGA CAT GGA GTC ATA GGC TCT G -3' 5'- CCA TTT TCC TTC TTG TGG AGC A -3' 5'- GCC ACC ACG CTC TTC TGT CT -3' 5'- TGA GGG TCT GGG CCA TAG AAC -3' PCR conditions were: 95 C for 3 s; 4 cycles of 95 C for 5 s, 6 C for 6 s; 1 stage of 95 C for 15 s, 6 C for 3 s, 95 C for 15 s. Other primers were as reported previously. 2-4
4 Figure S1. c-maf is not expressed in placenta and c-maf deficient placenta appeared normal (A-B) Immunofluorescence staining of c-maf in the lens section using anti-c-maf antibody (clone M-153, Santa Cruz Biotechnology) (A) and placental section (B). Green: c-maf, blue: Hoechst. The scale bar represents 2 µm in the upper panel of B. The other scale bar represents 1 µm. Representative pictures are shown. Note that c-maf localizes in the nuclei of lens because c-maf is a nuclear localizing transcription factor, whereas such localization is not observed in the placental section. (C) Relative c-maf mrna expression of placenta, limb and head. Total RNA obtained from 3 mg or less of these tissues were used for analyses. n = 4 per tissue from wild-type E13.5 animals. (D) Hematoxylin and eosin staining of placental sections from E13.5 c-maf +/+ and c-maf -/- animals. The scale bar represents 1 µm. Figure S2. Definitive erythropoiesis in fetal liver is impaired in c-maf -/- embryos (A) Typical flow cytometric profiles of c-maf +/+ and c-maf -/- fetal liver cells isolated from E13.5 embryos double labeled with a FITC-conjugated anti-cd71 monoclonal antibody (mab) and a APC-conjugated anti-ter-119 mab. Regions R1 to R5 are defined by a characteristic staining pattern of cells as indicated. The mean percentages for the populations in each region are shown in red. In c-maf -/- fetal liver, the percentages of R3, R4 and R5 were reduced, whereas the percentages of R1 and R2 were increased compared with c-maf +/+ fetal liver. (B) Comparison of c-maf +/+ and c-maf -/- fetal livers in the ratio of R1 to R5. represents c-maf +/+ ; represents c-maf -/-. n = 5 ~ 7 per group; *p <.5. (C) The ratio of annexin V positive cells from R1 to R5 was compared in c-maf +/+ and c-maf -/- fetal livers. represents c-maf +/+ ; represents c-maf -/-. n = 5 ~ 7 per group; *p <.5. Figure S3. Decreased mrna expression of βmajor/βminor globins in c-maf -/- fetal liver The Mac-1 negative fraction was isolated by Mac-1 magnetic beads. Analyses of mrna expression of mouse β-globin chains showed that mrna expression of βmajor/βminor globins was decreased,
5 whereas embryonic β-globin genes (εy-globin and βh1-globin) were maintained in the Mac-1 negative fraction of E13.5 c-maf -/- fetal liver cells. represents c-maf +/+ ; represents c-maf -/-. n = 7 per group. *p <.1. Expression of the c-maf +/+ fetal liver Mac-1 negative fraction was set to 1.. Data are presented as mean ± SEM. Figure S4. The in vitro erythroid colony formation of c-maf -/- fetal liver cells is not impaired (A) Hemoglobin levels in day 2 CFU-Es from c-maf +/+ and c-maf -/- E13.5 fetal liver cells are shown. The scale bar represents 2 µm. (B) Diameters of CFU-Es from c-maf +/+ and c-maf -/- E13.5 fetal liver cells are shown. represents c-maf +/+ ; represents c-maf -/-. Twenty-seven to twenty-nine colonies from 4 fetal livers per group were counted. Data are presented as mean ± SEM. p =.1. (C) Cytospun cells from day 2 CFU-Es (from E13.5 fetal liver cells in c-maf +/+ upper panel, and c-maf -/- ; lower panel) were stained with May-Grünwald Giemsa. Note enucleated red blood cells were observed in the cytospin picked from c-maf -/- CFU-Es. The scale bar represents 2 µm. Figure S5. Erythroid differentiation is not impaired in c-maf -/- fetal liver cells transplanted mice (A) Flow cytometric analysis of bone marrow cells prepared from femur of recipient mice that received c-maf +/+ or c-maf -/- fetal liver cells. Bone marrow cells were labeled with a FITC-conjugated anti-ter-119 monoclonal antibody (mab) and an APC-conjugated anti-cd44 mab. Regions I to V are defined by a characteristic staining pattern and forward scatter (FSC) intensity of cells as indicated 5. Representative profiles are shown. (B) Comparison of region I to region V showing ratios of bone marrow cells from mice transplanted with c-maf +/+ and c-maf -/- fetal liver cells. (C) The numbers of cells in region I to region V per femur are shown. Note that erythroid differentiation status in c-maf -/- fetal liver cells transplanted mice is comparable with that in the c-maf +/+ fetal liver cells transplanted control. represents bone marrow cells from mice transplanted with c-maf +/+ fetal liver cells; represents bone marrow cells from mice
6 transplanted with c-maf -/- fetal liver cells. n = 5 per group; FLC, fetal liver cells; Figure S6. VCAM-1 expression of bone marrow macrophages in c-maf -/- fetal liver cells transplanted mice is not decreased After 6-1 weeks following transplantation, the donor leukocytes (Left) in bone marrow cells and the donor macrophages in bone marrow macrophages (Right; Mac-1 positive cells) were analyzed. The chimerism of the mice reconstituted with c-maf -/- fetal liver cells was comparable with that of the mice reconstituted with c-maf +/+ fetal liver cells. Figure S7. c-maf -/- fetal liver macrophage mrna expression profiles at E13.5 mrna expression profiles of Rb and inflammatory cytokines. The population was enriched for the Mac-1-negative cells (open bar) and Mac-1-positive cells (gray bar). n = 7 per group. *p <.5. Data are presented as mean ± SEM. N.A. : not analyzed.
7 REFERENCES 1. Iavarone A, Emerson RK, Dai XM, et al. Retinoblastoma promotes definitive erythropoiesis by repressing Id2 in fetal liver macrophages. Nature. 24;432(72): Nakamura M, Hamada M, Hasegawa K, et al. c-maf is essential for the F4/8 expression in macrophages in vivo. Gene. 29;445(1-2): Atarashi K, Nishimura J, Shima T, et al. ATP drives lamina propria T(H)17 cell differentiation. Nature. 28;455(7214): Sankaran VG, Xu J, Ragoczy T, et al. Developmental and species-divergent globin switching are driven by BCL11A. Nature. 29;46(7259): Chen K, Liu J, Heck S, Chasis JA, An X, Mohandas N. Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis. Proc Natl Acad Sci U S A. 29;16:
8 igure S1 A c-maf Hoechest Merge B 1.5 Placenta Relative c-maf mrna expression Lens C D c-maf +/+ c-maf -/ Placenta Limb Head
9 Figure S2 A 1 4 c-maf +/+ c-maf -/- R2: 6.6 R3: 53.3 R2: 13.5 R3: B Cell Number ( x1 6 ) CD R1: TER c-maf +/+ * c-maf -/- R4: 12.3 R5:.6 R1 R2 R3 R4 R5 * * C Percentage of Apoptotic cells in each reagion (%) R1: R4: 8. R5:.3 R1 R2 R3 R4 R5 * *
10 Figure S3 Relative gene expression in Mac-1 negative fraction in E13.5 fetal liver cells c-maf+/+ c-maf-/- εy globin βh1 globin βmajor/βminor globins *
11 Figure S4 A B 3 c-maf -/- c-maf +/+ c-maf -/- c-maf +/+ C Diameter of CFU-E (μm) 2 1 c-maf +/+ c-maf -/-
12 Figure S5 A I IV c-maf +/+ FLC transplanted mice IV III II Count V III II I V TER-119 FITC FSC CD44 APC 1 4 I 1 4 IV III c-maf -/- FLC transplanted mice CD44 APC V IV III II Count V II I TER-119 FITC FSC CD44 APC B C Frequency of each region in TER-119 positive cells (%) c-maf +/+ FLC transplanted mice c-maf -/- FLC transplanted mice I II III IV V Cell number of each region per femur (x1 6 ) I II III IV V
13 Figure S6 c-maf +/+ FLC transplanted mice c-maf -/- FLC transplanted mice Percentage of doner leukocytes in BM cell (%) Percentage of doner macrophages in BM macrophages (%)
14 Figure S7 Relative gene expression Rb IL-6 Mac-1 positive Mac-1 negative c-maf +/+ c-maf -/- c-maf +/+ c-maf -/- * IL-1β * N.A. N.A. c-maf +/+ c-maf -/- IL-12β TNF α Relative gene expression c-maf +/+ c-maf cmaf -/- c-maf +/+ c-maf -/-
Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.
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CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'
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c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
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