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, For all experiments, littermates of the same sex were randomly assigned to experimental groups. Mice were given ad libitum access to food and water with a 12 hours light/ 12 hours dark cycle. For experiments involving mouse embryos, Tph1 wild-type, heterozygous, and homozygous males (10-12 weeks old) bred to Tph1 wild-type, heterozygous, and homozygous females (10-12 weeks old). For in vivo experiments involving fluoxetine and sub-lethal irradiation, WT mice and Tph1 +/À were used (males and females 8-10 weeks old and ?8 months old). For bone marrow transplantation experiments, WT females and Tph1 À/À 8-10 week's old female mice were used. For murine erythroid in vitro cell culture and immunophenotyping of murine erythroid precursors, EXPERIMENTAL MODEL AND SUBJECT DETAILS Mouse models For all experiments animals were housed 5 mice/cage. Tph1 À/À mice were generated as described, 2003.
, Tph1 À/À , Tph1 +/À and WT animals (males and females) were derived from pure C57BL/6J genetic backgrounds. For some experiments, C57/bl6 mice (males and females) were purchased from Janvier Labs (CS 4105, 2003.
, The day at which the vaginal mating plug was seen was taken as E0.5. Mice were sacrificed by cervical dislocation; the fetal livers were removed from E10.5 to E18.5 and stained with appropriate antibodies. For BrdU incorporation, pregnant females were injected i.p. a dose of BrdU (1 mg) dissolved in PBS 2 hours before sacrifice. For cell cycle analysis, BrdU incorporation level in CD71 + /c-Kit +/À /TER119 -cells was assessed using a BrdU flow kit according to the manufacturer's instructions (BD PharMingen). For RT-qPCR analysis, CD71 + /c-Kit +/À /TER119 -cells were isolated from fetal livers using a FACS-AriaII cell sorter (BD Biosciences), Embryos Embryos were obtained from crosses between Tph1 wild-type, heterozygous, and homozygous males bred to Tph1 wild-type, heterozygous, and homozygous females (8-10 weeks old)
, Histological analysis For histology, tissues were fixed in 10% formalin overnight, embedded in paraffin blocks, and sectioned (5 mM). Sections were stained with Prussian blue. Smears and culture of pure pro-erythroblasts cells (c-Kit +/À /CD71 + /TER119 -) derived from bone marrow of WT and Tph1 +/À aged mice (males and females > 8 months old) were stained with May-Gruenwald Giemsa for analysis. Patients Low and int-1 MDS patients (n = 71) were enrolled after they gave their informed consent according to the recommendations of the institutional ethics committee (CPP Ile-de-France X). Peripheral blood plasma (n = 71) and bone marrow RNA (n = 27) were obtained at diagnosis for tryptophan and kynurenine dosages, and for RNA-sequencing, respectively. Plasmas and bone marrows from agematched subjects were used as controls. For 5-HT measurements, whole blood was collected from MDS patients (n = 15; 9 men, 6 women) and aged-matched controls (n = 14) on sodium-citrate tubes. For the RNA seq experiment (Figure 1A), n = 27; 22 men, 5 women. For the IDO, Hb, Trp measurement (Figures 1B-1E) n = 71, Sub-lethal irradiation was induced by submitting WT or Tph1 +/À mice to 1.09 gy during 4 minutes (WT C57/bl6 Males 8-10 weeks old purchased form Janvier labs for Figures 6D and 6E), 2015.
, from fetal livers or flushed form femur/tibia were grown in StemPro-34 serum-free stem cell expansion medium supplemented with human recombinant EPO (1 U/mL), murine recombinant stem cell factor (100 ng/mL) (Immunotools), Cell Reports, vol.26, pp.3246-3256, 2019.
, mM) (Tocris Bio-Science) and 1-methyl-tryptophan (1-MT) (Sigma) was added to the proliferation/differentiation medium. Proliferation was monitored by cell counts. Treatment of CD71 + /c-Kit +/À /TER119 -mouse bone marrow cells for 2-4 hours with STAT5 inhibitors (10uM pimozide, Sigma), followed by RT-qPCR analysis for Tph1 mRNA. Apoptosis was measured using APClabeled annexin V (BD PharMingen) following staining with cell surface markers, some conditions, 5-HT HCl (1 mM; Sigma), 52954.
, minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. Cells were then incubated in 1X PBS/10% normal goat serum followed by the antibody (ab52954, 1:100 dilution) for 30 minutes at room temperature. The secondary antibody used was DyLight 488 goat antirabbit IgG (ab96899) at 1:500 dilution for 30 minutes at room temperature. Isotype control antibody was rabbit IgG (monoclonal) used under the same conditions, ) according to the manufacturer's protocol. Briefly, CD71 + /c-Kit +/À /TER119 -were fixed with 80% methanol
, BrdU incorporation level in CD71 + /c-Kit +/À /TER119 -cells was assessed using a BrdU flow kit according to the manufacturer's instructions (BD PharMingen). For RT-qPCR analysis, CD71 + /c-Kit +/À /TER119 -cells were isolated from bone marrow using a FACS-AriaII cell sorter (BD Biosciences). Human cord blood Erythroid in Vitro Cell Culture Erythroid cells were generated from CD34 + cord blood progenitor cells in serum-free medium
, The study was performed according to the Helsinki Declaration with the approval from the ethics committee of our institution (Comité de Protection des personnes (CPP) ''Ile de France II''). All patients gave written informed consent. Cord blood samples were collected from umbilical veins following normal full-term vaginal deliveries and processed within 24 hours. Immunophenotyping of Murine Erythroid Precursors Cells from fetal livers or BM cells flushed from femur and tibia were resuspended in Hank's buffered saline before being passed through a 100-mM strainer. Cells were washed, counted, and immunostained at room T in PBS with PE-conjugated anti-TER119, FITC-conjugated anti-CD71 and APC-conjugated anti-CD117 (c-Kit) (BD PharMingen and BioLegend) antibodies for 20 min and analyzed on a FACS Canto, ng/ml) + stem cell factor (SCF; 50 ng/ml) as previously described in Zermati, 2000.
, sacrificed by cervical dislocation and the femur/tibia were removed, weighed, and sonicated for 5 s in10 volumes (vol/wt) of 0.1 N perchloric acid 0.05% disodium EDTA0.05% sodium metabisulfite. 5-HT was extracted, and 10-l samples were injected onto a Beckman Ultrasphere 5-m IP column (Beckman)
, Total RNA was extracted from fetal liver cells, bone marrow cells and CD34 + /CD36 + cord blood cells using the RNeasy Kit (QIAGEN)
, Two biological replicates were used for each condition. Data were analyzed by StepOne Plus RT PCR software v2.1 and Microsoft excel. b-actin, GAPDH and 18S transcript levels were used for normalization of each target ( = DCT), Reverse transcription was performed using iScript Reverse Transcription Supermix for RT-qPCR (BioRad)
, CA) and sequenced on an Illumina HiSeq 2500 platform using a 100-bp paired-end sequencing strategy. An average depth of global sequence coverage of 114 million and a median coverage of 112 million was attained. For analysis, TopHat (v2.0.6) was used to align the reads against the human reference genome Hg19 RefSeq (RNA sequences, GRCh37) downloaded from the UCSC Genome Browser, RNA-sequencing Libraries were constructed using the TruSeq Stranded mRNA Sample Preparation Kit