Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 1 1 SUPPLEMENTAL INFORMATIONS 2 Supplemental Figures with legends 3 A WT young B WT aged ** 6 * 6 4 4 ns ns ns fold LT-HSC ns 2 fold LT-HSC 2 ns ns ns ns ns ns 0 0 MPP CMP GMP MEP liver MPP CMP GMP MEP liver LT-HSC ST-HSC LT-HSC ST-HSC C WT young D *** ns ns 1.5 1.0 ns ns 1.0 ** 0.5 fold HSC 0.5 fold of young 0.0 0.0 Young Aged Old HSC MPP CMP GMP MEP Figure S1. Pon2 mRNA expression in different bone marrow cells of WT mice A and B: LT-, ST-HSCs, MPPs, CMPs, GMPs, MEPs and liver cells were obtained from young (2-3 months) and aged (>9 months) WT mice. Pon2-, Gapdh- and Actb mRNA expression was analyzed by qRT-PCR. Pon2 mRNA expression was normalized to Gapdh and Actb-mRNA expression. The relative Pon2 mRNA expression in LT-HSCs from young or aged WT mice was set to 1. Shown are the mean ± SEM of n=3-6 experiments using 2- 6 mice per group (**p < 0.01, *p < 0.05, ns not significant vs. LS-HSC cells; 1-way Anova with Dunnett's multiple comparisons test). C: RNA-Seq reads from bioproject PRJNA631793 and PRJNA665066 were analyzed for Pon2 gene expression. The values (rpkm) of the HSC of young mice were set as 1fold. Shown are the mean ± SEM (*** p < 0.001 ** p < 0.01, ns not significant vs HSC; 1-way Anova with Dunnett’s multiple comparisons test). D: RNA-Seq reads from bioproject PRJNA524895, PRJNA528500 and PRJNA635499 and Microarray-Data from GSE76276; were analyzed for Pon2 gene expression. The values (rpkm or normalized intensities) of the young mice were set as 100 %. Shown are the Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 2 mean ± SEM (ns not significant vs. LT-HSC cells form young mice; 1-way Anova with Dunnett’s multiple comparisons test). 4 5 Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 3 A B ns 200 ns *** 150 *** *** *** 100 50 0 PON2 mRNA expression (% of WT/WT) PON2 mRNA WT PON2-/- PON2-/- WT recipient WT PON2-/- WT PON2-/- donor C D E ns ns * ns ns ns ns ns 5 ns ns 6×10 5 ns WBMC) 3×10 WBMC) 8 ns 8 WBMC) * ns 8 ns ns ns ns 5 4×10 2×105 5 2×10 1×105 0 0 0 WT PON2-/- PON2-/- WT -/- -/- -/- -/- recipient recipient WT PON2 PON2 WT recipient WT PON2 PON2 WT -/- -/- donor WT PON2 WT PON2 -/- -/- -/- -/- donor WT PON2 WT PON2 MPPs (abs. cell number per 2.5 10 donor WT PON2 WT PON2 LT-HSCs (abs. cell number per 2.5 10 ST-HSCs (abs. cell number per 2.5 10 ST-HSCs F G ns H ns ns ns ns ns ns ns ** ns 6 ns 5 6 1.5×10 8×10 * ns 1.5×10 ns WBMC) WBMC) WBMC) 8 8 ns 8 ** ns ns 6×105 1.0×106 1.0×106 4×105 5.0×105 5.0×105 2×105 0.0 0 0.0 -/- -/- -/- -/- recipient WT PON2 PON2 WT recipient WT PON2 PON2 WT recipient WT PON2-/- PON2-/- WT -/- -/- -/- -/- MEPs (abs. cell number per 2.5 10 CMPs (abs. cell number per 2.5 10 donor WT PON2 WT PON2 donor WT PON2 WT PON2 GMPs (abs. cell number per 2.5 10 donor WT PON2-/- WT PON2-/- 6 7 8 Figure S2. In young Pon2-/- mice, reciprocal BM transplantation reveals cell intrinsic as 9 well as extrinsic pHenotypes. (A) Experimental scheme for reciprocal transplantation. (B) 10 Verification of engraftment of hematopoietic stem cells and reconstitution of hematopoiesis by 11 donor stem cells in recipient mice using qRT-PCR based analysis of Pon2 mRNA expression 12 in isolated blood cells of transplanted animals (n=8-10). Results are presented as percentage 13 of WT/WT and normalized to Gapd- and Actb-mRNA expression. (C) Percentage of LT-, (D) 14 ST-HSCs, (E) MPPs, (F) CMPs, (G) MEPs and (H) GMPs in WBMCs of BM-chimeras and 15 transplantation controls (n=10-13). Box and whiskers; Whiskers: 10-90 percentile. * p≤0.05, ** 16 p≤0.01, ns not significant; One-way Anova with Tukey's multiple comparisons test. Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 4 A B young aged WT WT PON2-/- PON2-/- counts counts gamma-H2AX FITC-A gamma-H2AX FITC-A 17 18 19 Figure S3. Pon2-/- BMCs show no enhanced amount of DNA double-stand breaks in LSK 20 cells. Overlay of histograms showing the amount of DNA double-strand breaks in WT (green) 21 and Pon2-/- (red) LSK cells, determined by anti-gamma-H2AX staining using BMCs from (A) 22 young or (B) aged animals. One representative of 4 experiments is shown. 23 24 Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 5 25 G0 Colony forming units Colony forming units A G1 LSK B G0 LT-HSC's C D S,M,G2 G1 150 30 S,M,G2 WT ns ns PON2-/- ns ns ns 100 ns ns cells 100 4 ns ns ns 100 ns ns cells 20 4 ns 75 ns ns ns ns ns 75 50 50 10 50 Colonies / 10 total Colonies / 10 Cell cycle phase (%) 25 Cell cycle phase (%) 25 0 0 WT PON2-/- -M -G -GM -GEMM -E 0 0 WT PON2-/- WT PON2-/- F Homing E 50 45 ns 40 35 30 25 20 15 10 % DiI pos. cells 5 0 WT-BM PON2-/--BM 26 27 28 29 30 Figure S4. Bone marrow cells of young Pon2-/- and WT mice show no differences in cell 31 cycle status, colony forming ability and Homing. (A/B) Cell-cycle analysis of LSK (A) and 32 LT-HSCs (B) of young WT and Pon2-/- mice as percentage within each cell-cycle phase (n=6). 33 (C) Colony-forming unit assay (CFU): Total numbers of counted colonies and (D) observed 34 CFU-M (colony-forming unit macrophage), CFU-G (colony-forming unit granulocyte), CFU-GM 35 (colony-forming unit granulocyte/macrophage), CFU-GEMM (colony-forming unit granulocyte/ 36 erythroid/macrophage/megakaryocyte) and CFU-E (colony-forming unit erythroid) colonies 37 from 3x104 WT and Pon2-/- BMC’s, 10-12 days after plating (n=12). (E) Experimental scheme 38 of homing experiment. (F) Percentage of DiI positive WT and Pon2-/- BMCs isolated by flushing 39 tibial and femoral bones of lethally irradiated WT recipient mice 48 hours after transplantation 40 (n=10); ns not significant vs. WT, two tailed unpaired t-test. 41 42 Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 6 43 A LSK-fraction: LT-HSCs: lin-, Sca1+, ckit+, CD150+, CD135- ST-HSCs: lin-, Sca1+, ckit+, CD150-, CD135- MPPs: lin-, Sca1+, ckit+, CD150-, CD135+ B Oligopotent progenitors – myeloid lineage CMPs: lin-, Sca1-, ckit+, CD16/32-, CD34+ GMPs: lin-, Sca1-, ckit+, CD16/32+, CD34+ MEPs: lin-, Sca1-, ckit+, CD16/32-, CD34- C Apoptosis Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 7 D Competitive transplantation Donor Recipient E Cell cycle A - SSC Count Hoechst 33342 Hoechst FSC-A Hoechst 33342 Hoechst 33342 A LSK A - - A - Cy5 - PE ckit lin Alexa 700 lin Alexa CD150 PE FSC-A Sca APC-Cy7-A CD48 PE-Cy7-A Cell cycle LT-HSCs Cell cycle LSK cells Gates Q1 and Q 1-1 represent cells in G1- 67 67 - - phase, Gates Q2 and Q2-1 represent cells Ki Ki in the G2-, S- & M-phases and Gates Q3 and. Q3-1 represent cells Zellen the G0- phase. Hoechst 33342 Hoechst 33342 F Homing DiI-A Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 8 G Total ROS-Level H DNA-damage I Erythrocyte Precursors Figure S5. Gating strategies. Gating strategy for the measurement of (A) HSPC subpopulations within the LSK fraction of BM, (B) Oligopotent progenitors in the BM (C) apoptotic rate of LT- and ST-HSCs, (D) CD45.1 or CD45.2 cells in competitive transplantation experiments, (E) LSK cells and LT-HSCs in different stages of the cell cycle, (F) Dil-positive cells in homing experiments, (G) total ROS-Level of LT-, ST-HSCs and MPP, (H) DNA-damage experiments (I) Erythrocyte Precursor cells. 44 FACSDiva Version 6.1.3 FACSDiva Version 6.1.3 FACSDiva Version 6.1.3 FACSDiva Version 6.1.3 Spiecker et al. The redox enzyme paraoxonase-2 modulates hematopoiesis 9 A B C D E F 45 Figure S6. Representative Histograms sHowing the DCF-DA data of LT- and ST-HSC as 46 wellH2DCF-DA as MPP BM isolated from young WT and Pon2PageH2DCF-DA-/ -2 mice. of 7 BM Printed on: Wed Feb 10, 2016 10:51:25Page CET 3 of 7 Printed on: Wed Feb 10, 2016 10:51:30 CET 47 Total ROS-level in LT- (A/B), ST-HSCs (C/D) and MPPs (E/F) of young (2-3 months) WT H2DCF-DA BM Page 4 of 7 Printed on: Wed Feb 10, 2016 10:51:36 CET 48 (A/C/E) and Pon2-/- (B/D/E) mice stained with cell specific markers (LT-HSCs: Lin-, Sca1+, 49 ckit+, CD135-, CD150+; ST-HSCs: Lin-, Sca1+, ckit+, CD135-, CD150-; MPPs: Lin-, Sca1+, 50 ckit+,H2DCF-DA CD135+, BM CD150-) and H2DCF-DA, analyzedPage 1 ofby 7 FACS (n=12Printed-13). on: Wed Feb 10, 2016 10:51:20 CET 51 52 Spiecker et al.