050K100K150K200K250K 0 50K 100K 150K 200K 250K 0.47 0.6 050K100K150K200K250K 0 50K 100K 150K 200K 250K 0.65 0.15 4ug/ml hoechst 5ug/ml hoechst LINEAGE.

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050K100K150K200K250K 0 50K 100K 150K 200K 250K K100K150K200K250K 0 50K 100K 150K 200K 250K ug/ml hoechst 5ug/ml hoechst LINEAGE DEPLETED MOUSE BONE MARROW LABELLED WITH HOECHST AT CONCENTRATIONS SHOWN: 4UG/ML RESOLVES A GREATER PROPORTION OF ‘SP’ CELLS

x x x x ug/ml hoechst 5ug/ml hoechst LINEAGE DEPLETED MOUSE BONE MARROW SHOWING SP CELLS: HIGHLY ENRICHED FOR KLS

050K100K150K200K250K 0 50K 100K 150K 200K 250K K100K150K200K250K FSC-A 0 50K 100K 150K 200K 250K SSC-A 050K100K150K200K250K FSC-A 0 50K 100K 150K 200K 250K SSC-A PI GATING CLEANS UP FSC SSC PLOT - undepleted bone marrow sample -PI strongly excited by UV laser, emits in same filter as H Red signal -Much stronger than hoechst, but doesn’t stain RBCs 1. BEFORE GATING 2. PI NEGATIVE GATE 3. AFTER GATING

050K100K150K200K250K 0 50K 100K 150K 200K 250K 050K100K150K200K250K 0 50K 100K 150K 200K 250K 050K100K150K200K250K 0 50K 100K 150K 200K 250K SP ANALYSIS - NO PI SP ANALYSIS - PI INCLUDED 050K100K150K200K250K 0 50K 100K 150K 200K 250K 0.6

VERAPIMIL BLOCKS SP POPULATION - verapimil blocks transporter responsible for pumping hoechst out of sp cells 050K100K150K200K250K 0 50K 100K 150K 200K 250K 9.16e-3 050K100K150K200K250K 0 50K 100K 150K 200K 250K PI NEGATIVE NOTE THAT PI NEGATIVE EVENTSS REPRESENT 45.5% OF EVENTS IN SAMPLE WITHOUT TREATMENT - THIS DROPS TO 6% IN VERAPRIMIL TREATMENT SUGGESTING GENERAL TOXICITY OF THIS DRUB NON LINEAGE DEPLETED BONE MARROW STAINED WITH HOECHST ALONE (LEFT) OR HOECHST IN THE PRESENCE OF VERAPIMIL (RIGHT)