Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards

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1 Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards incubated in 100 % ethanol overnight at 4 C and embedded in paraffin. 5 µm sections were dewaxed, rehydrated in descending alcohol row, stained in toluidin blue solution (0,3 % in aqua bidest) for 10 min, dehydrated in an ascending alcohol row, cleared in Xylol and mounted with Entellan. Slides were analyzed in a blinded way using with an Axiovert 200M microscope (Carl Zeiss) equipped with AxioCam MR3 (Carl Zeiss) and AxioVision software (version 2.2.5).

2 gene name forward primer reverse primer designed by Elane tgg agg tca ttt ctg tgg tg ctg cac tga ccg gaa att tag Roche Ltf ggg caa gtg cgg ttt agt t cca ttg ctt ttg gag gat tt Roche Mmp9 acg aca tag acg gca tcc a gct gtg gtt cag ttg tgg tg Roche Mpo gga agg aga cct aga ggt tgg tag cac agg aag gcc aat g Roche Ngp gcc taa aga ctg cga ctt cc tga aga att tcc ctg tgc aa Roche Actb caa cga gcg gtt ccg atg gcc aca gga ttc cat acc ca RTprimerDB Btk ggc cat caa gat gat cag aga gct tct cat ggg aaa gat tca Roche Cebpa aaa caa cgc aac gtg gag a gcg gtc att gtc act ggt c Roche Cebpb tga tgc aat ccg gat caa cac gtg tgt tgc gtc agt c Roche Sfpi1 tct tct gca cgg gga gac ag gga cga gaa ctg gaa ggt acc Roche Gata1 gaa tcc tct gca tca aca agc ggg caa ggg ttc tga ggt Roche Csf2ra aga gcc agg aag cac acc cag tgc ttc atc ctc gtg tc GeneScript Csf3r tat gct agg gtc cag cga gt ggg agg ctc caa ttt cac a Roche Csf1r gtc atg tct ctg ctg gtg ct tgc ctt cgt atc tct cga tg GeneScript Gapdh gac ttc aac agc aac tcc cac tcc acc acc ctg ttg ctg ta RTprimerDB Hprt cct aag atg agc gca agt tga a cca cag gac tag aac acc tgc taa RTprimerDB Table S1. Depicted are sequences of the used primers as well as the company by which the primer was designed

3 Figure S1. Frequencies of myeloid cell populations are altered in spleens of Btk-deficient mice Spleens of wildtype (wt) and Btk-deficient (Btk-ko) mice were analyzed for the frequency of cell subpopulations indicated. (A-F) Cell suspensions of spleens from wildtype and Btk-deficient (Btk-ko) mice were (A) counted and stained for (B) B220, (C) CD11b, (D) CD11b/CD11c, (E) Gr-1 (F) CD11b/F4/80. Data presented are mean values (±SD). (*) P 0.05; (**) P 0.005; (***) P n represents the number of biological replicates. Figure S2. Frequencies of myeloid and erythroid cell populations in the blood and bone marrow of Xid mice Blood (A and B) from wildtype (CBA/J) and Xid/J mice were analyzed using an Animal Blood Counter for differential white blood cell counts. Frequencies (A) as well as cell numbers per volume (B) of lymphocytes, neutrophilic granulocytes and monocytes are shown. (C) The bone marrow cells of femurs of wildtype and Xid mice were stained for the expression analysis of surface markers CD11b (myeloid cells), Ter119 (erythrocytes) and B220 (lymphocytes). (D) The myeloid compartment was further analyzed by the expression of CD11b + Gr-1 + (granulocytes) and CD11b + Gr-1(monocytes). The frequency of cell populations was re-calculated for defined cell numbers per femur (E and F). Data presented are the mean values (±SD). (*) P n represents the number of biological replicates. Figure S3. GMP deficient for Btk exhibit a decreased differentiation potential and drive myeloid development towards the granulocytic lineage upon TLR stimulation 500 GMP sorted out of individual wildtype and Btk-deficient (Btk-ko) mice were seeded in methylcellulose media supplemented with SCF, IL3, Flit-3 ligand and LPS (A to E) or SCF, IL3, Flit-3 ligand and Pam3CSK4 (F to H). The experiments were performed in triplicates. (A and F) After 6 days in culture colony-forming units were counted. (B and G) The cell number per CFU was calculated by determining the number of cells developing from 500 seeded GMP per CFU. (C and H) Differentiated cells were analyzed by flow cytometry for the surface marker CD11b, Gr-1 and F4/80. Data presented are the mean (±SD) of cell numbers positive either for the surface marker CD11b + /Gr-1 - /F4/80 - (undifferentiated cells), for CD11b + /Gr-1 + (granulocytes), or positive for CD11b + /F4/80 + (monocytes). (D) Twenty individual CFU per biological replicate were processed for cytospins and Pappenheim staining and analyzed for the cell content by morphology. A CFU was considered as CFU-M if more than 90% of the cells were macrophages, as CFU-G (here not detected) if more than 90% of cells were neutrophils, and CFU-GM if the content of macrophages and granulocytes was in between 20 to 80%. (E) One CFU with a representative size generated from wildtype or Btk-deficient (Btk-ko) GMP was photographed and is presented. Scale bars represent 200 µm. Data presented are the mean values (±SD). (**) P 0.005, (**) P n represents the number of biological replicates. Figure S4. The numbers of granules are reduced in Btk deficient neutrophils Electron microscopy was performed using wildtype or Btk-deficient neutrophils. Representative images of each genotype are shown. Figure S5. Neither LPS nor G-CSF-treatment rescues the developmental phenotype of myeloid precursors or neutrophils in Btk-deficient mice

4 (A) Bone marrow obtained from wildtype and Btk-deficient mice were treated in vitro with G- CSF (Promokine; 25 ng/ml), GM-CSF (Miltenyi; 25 ng/ml), LPS (Invivogen; 10 µg/ml), or left untreated. The development of CD11b + /Gr-1 + granulocytes was analyzed by FACS after 72 hours of culture. (B to I) Animals were left untreated or injected intravenously with LPS, G-CSF, or GM-CSF in PBS (25 µg/kg body weight each). 36 hours later the blood of mice was taken and analyzed for relative and absolute numbers of granulocytes (B and C). Afterwards, mice were sacrificed and the bone marrow was analyzed for the absolute numbers of granulocytes per femur (D), their maturation status according the expression level of CD11b and Gr-1 (E). Additionally, changes in the myeloid precursor cell compartments were analyzed by FACS (F). GMP were isolated by FACS and analyzed by RT-PCR for the expression of C/EBPα (Cebpa), gelatinase (Mmp9) and Elastase (Elane) relative to the expression of Hprt1.

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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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