7-amino actinomycin D (7ADD) was added to all samples 10 minutes prior to analysis on the flow cytometer in order to gate 7AAD viable cells.

Transcription

1 Antibody staining for Ho uptake analyses For HSC staining, 10 7 BM cells from Ho perfused mice were stained with biotinylated lineage antibodies (CD3, CD5, B220, CD11b, Gr-1, CD41, Ter119), anti Sca-1-PECY7, anti KIT-APC, CD48-FITC and CD150-PE. After 40 minutes on ice, cells were further stained with streptavidin-apccy7. For HSC staining with CD34 and anti-flt3 antibodies, BM cells were stained exactly as above except that CD34-FITC and anti FLT3-PE were used instead. For myeloid progenitors, antibody used were biotinylated lineage antibodies (CD3, CD5, B220, Gr-1, Ter119) and biotinylated anti-il7rα together with Sca-1-PECY7, KIT-APC, CD34-FITC and CD16/32-PE followed by streptavidin-apccy7. Common myeloid progenitors (CMP) were defined as Lin IL7Rα Sca-1 KIT + CD34 + CD16/32, granulocyte monocyte progenitors (GMP) as CD34 + CD16/32 +, and megakaryocyte erythrocyte progenitors (MEP) as CD34 CD16/32 within the same Lin IL7Rα Sca-1 KIT + gate. For stromal cells, endosteal cells were stained with FITC-conjugated lineage antibodies (CD3, CD5, B220, CD11b, Gr-1, Ter119), CD45-APCCy7, CD31-APC, CD51-PE, and anti Sca-1- PECY7. 7-amino actinomycin D (7ADD) was added to all samples 10 minutes prior to analysis on the flow cytometer in order to gate 7AAD viable cells. Two million events per sample were acquired or sorted on a FACS Aria cell sorter (BD Biosciences) equipped with 407nm, 488nm and 633nm lasers. Hoechst33342 blue and red fluorescences were analyzed using 450/40nm and 610/20nm filters respectively following excitation with the 407nm violet laser. Data were analyzed following compensation with single color controls using the FloJo software (Tree Star, Ashland, OR) on a MacIntosh platform (Apple). BrdU label-retention analyses Mice were administered BrdU for 14 days in drinking water (0.5mg/mL), then allowed to rest (chase) for 70 days. Ho was administered intravenously precisely 10 and again 5 minutes before sacrifice. Immediately after sacrifice, femurs, tibias and pelvis were immediately removed and crushed in ice cold PBS containing 2% FCS, 5µM reserpine, 50 µm verapamil to quicky release BM leukocytes which were stained with biotinylated lineage antibodies (CD3, CD5, B220, CD11b, Gr-1, CD41, Ter119) and SAV-PercPCY5.5, anti Sca1-PECY7, anti KIT-APC-H7, CD48-FITC, CD150-PE. From each individual mouse, Ho neg and Ho med BM cells within the Lin KIT + gate were sorted, then fixed and stained for BrdU incorporation using reagents and instructions from the BrdU-APC flow kit from BD Pharmingen (Caltalog# ). Fluorescence and BrdU incorporation were analyzed on a CyAn ADP7c flow cytometer equipped with 405nm, 488nm and 642nm lasers. The percentage of cells that retained BrdU within the LSK CD41 CD48 CD150 + Ho neg and Ho med populations was measured. Cell cycle analyses BM leukocytes were enriched for KIT + cells by MACS and subsequently stained with biotinylated lineage antibodies, anti Sca-1-PECY7, anti KIT-APC and CD48-PE followed by

2 streptavidin-alexafluor700. Washed cells were then fixed and permabilized using the FIX AND PERM kit from Caltag. FITC-conjugated mouse anti-human Ki67 (or FITC-conjugated isotype control mouse IgG1) was added to the permabilization buffer for 30 minutes. After washing, cells were incubated with 1µg/mL RNAse A and 0.5µg/mL DAPI for 15 minutes prior to analysis. Data were acquired on a LSRII flow cytometer equipped with 355nm, 488nm and 633nm lasers. Immunohistochemistry and enumeration of blood vessels in the BM Femurs from mobilized and non-mobilized mice were immediately incubated in zinc fixation solution, then decalcified, paraffin embedded, and sections stained for CD31 [Vanderkerken, 2005 #1974]. To enumerate blood vessels, snapshots of adjacent fields across each section (approx 10 per section) at 200 magnification were taken. For each snapshot, blood vessels were counted and the area of BM determined using Image J software. The total number of blood vessels and the total BM area from all the snapshots for each section was calculated. Total blood vessel number was normalized to the BM area on each section.

3 Figure S1. Delineation of Ho neg, Ho med and Ho bright gates For each individual experiment, the Ho neg, Ho med and Ho bright gates were defined by analyzing Ho blue and Ho red fluorescence on a logarithmic scale before running antibody stained samples. Viable 7AAD-negative blood leukocytes from a mouse injected retroorbitally with Ho 10 and 5 minutes before sacrifice (panel A) were used to define the Ho bright gate, and viable BM leukocytes from a control mouse that was not injected with Ho (panel B) were used to define the Ho neg gate. The Ho med gate was drawn between the Ho neg and Ho bright gates. Panel C is an example of Ho staining obtained from BM leukocytes from a Ho injected mouse. Note in panel C that unlike blood leukocytes (A), Ho uptake in BM leukocytes spreads over the three Ho gates. Figure S2. Absence of detectable Ho efflux from BM HSC and multipotent progenitor cells stained with Ho ex vivo for 10 minutes BM cells from non-perfused C57BL/6 mice were isolated and stained ex vivo for 10 minutes at 37 C with increasing Ho concentrations in the presence or absence of verapamil and reserpine as indicated on the top row. Cells were then immediately washed and stained on ice in the presence of verapamil and reserpine as described in figure 1. Cells were gated for viable Lin CD41 Sca- 1 KIT + lineage restricted progenitors, Lin CD41 Sca-1 + KIT + CD150 short-term reconstituting multipotent progenitors and Lin CD41 Sca-1 + KIT + CD48 CD150 + HSC as described in figure 1 and Ho uptake was measured on dot plots of Ho blue fluorescence versus Ho red fluorescence at each Ho concentration in the absence or presence of pump inhibibitors. (A) Mean fluorescence intensities (MFI) in the Ho blue channel were plotted versus Ho concentration and linear regression calculated for the 3 above mentioned populations. Differences in regression slopes were not significant. (B) Ho fluorescence dot-plots for Lin CD41 Sca-1 + KIT + CD48 CD150 + phenotypic HSC labeled in vitro with increasing concentrations of Ho in the presence or absence of verapamil and reserpine. Lin CD41 Sca-1 + KIT + CD150 short-term reconstituting multipotent progenitors and lineage-restricted Lin-Sca1- KIT + progenitors showed similar pattern (not shown). Figure S3. Cycling of BM HSC and HPC BM leukocytes were harvested from four C57BL/6 mice. Following enrichment of BM KIT + cells by MACS, cells were stained for Lin, Sca-1, KIT and CD48 before fixation and permeabilization. Cells were then stained for the nuclear antigen Ki67 and DNA content with DAPI. A: Sca-1 and CD48 expression on gated Lin KIT + cells. B: Cells cycle analysis according to Ki67 expression and DNA content on Lin KIT + Sca-1 (L K + S ), Lin KIT + Sca-1 + CD48 + (L K + S CD48 + ) and Lin KIT + Sca-1 + CD48 (L K + S CD48 ) cells gated in panel A. C: Proportion of gated HPC in G0, G1 and S/G2/M phases. These data are average±sd from 4 different mice. Figure S4. Ho uptake does not interfere with engraftment BM leukocytes were harvested from B6.SJL CD mice and incubated for 10 minutes at 37 C with 200µM Ho (black circles) or with saline (white circles). 200,000 of these leukocytes were mixed with equal number of competing BM leukocytes from C57BL/6 mice and transplanted into lethally irradiated C57BL/6 recipients. CD45.1 chimerism was measured in total leukocytes, myeloid, B and T cells 16 weeks post-transplant. Each dot represents an individual recipient and bars are average for each group.

4 A Setting Ho bright gate B Setting Ho neg gate C Hoechst Blue Hoechst red Blood leukocytes Ho perfused BM leukocytes No Ho BM leukocytes Ho perfused Figure S1

5 A LSK CD41 - CD48 - CD150 + Ho blue, MFI no VR V+R Ho, µm LSK CD41 - CD150 - B 10 min Ho at 37 C in vitro With Verapamil+reserpine Without Ho 0µM Ho 5µM Ho 5µM Ho blue, MFI Ho blue, MFI no VR V+R Ho, µm Lin - Sca1 - KIT Ho, µm Hoechst Blue Ho 10µM Ho 10µM Ho 20µM Ho 20µM Ho 50µM Ho 50µM Ho 200µM Ho 200µM Hoechst red LSK CD41 - CD48 - CD150 + Figure S2

6 CD L-K+S- L-K+S+ CD Sca-1 L-K+S+ CD48- % of population in cell cycle phase L- K+ S- 40 L- K+ S+ CD L- K+ S+ CD G0 G1 S-G2-M L-K+S- L-K+S+ CD48+ L-K+S+ CD48- G1 S/G2/M G1 S/G2/M G1 S/G2/M Ki67 G0 G0 G0 DAPI (DNA) Figure S3

7 CD cells % of total blood leukocytes Total CD45 + leukocytes CD11b + myeloid cells B220 + B cells CD3 + T cells Sal Ho Sal Ho Sal Ho Sal Ho Figure S4