Figure 6.
PLCG1 is dispensable for normal HSC function. (A) Experimental protocol for investigation of steady-state hematopoiesis. (B) White blood count (WBC), hemoglobin (HGB), and platelets (PLT) following genetic inactivation of Plcg1 (Plcg1−/−, n = 6) for 16 weeks of steady-state hematopoiesis, compared with Plcg1+/+ controls (n = 14). (C) Immunophenotypic quantification of mature myeloid (Gr-1 Mac-1; F4/80), B-lymphoid (B220; CD19), and T-lymphoid (CD3) bone marrow cells (Plcg1+/+, n = 10; Plcg1−/−, n = 6). (D) Immunophenotypic quantification of stem and progenitor cell abundance, specifically of hematopoietic stem cells (HSC: CD150+ CD48− L−S+K+) and multipotent progenitors (MPP: CD150 low, CD48+ L−S+K+) (Plcg1+/+, n = 10; Plcg1−/−, n = 6). (E) Protocol for assessing impact of Plcg1 loss on LT-HSC function by serial transplantation. (F) Peripheral blood chimerism of primary recipient mice (Plcg1+/+, n = 6; Plcg1−/−, n = 11); shown are 2 independent cohorts. (G) Immunophenotypic quantification of mature myeloid (Gr-1), B-lymphoid (B220; CD19), and T-lymphoid (CD3) bone marrow cells (Plcg1+/+, n = 6; Plcg1−/−, n = 6) from primary recipients. (H) Immunophenotypic quantification of stem and progenitor cell abundance, specifically of hematopoietic stem cells (HSC: CD150+ CD48− L−S+K+) and multipotent progenitors (MPP: CD150 low, CD48+ L−S+K+) (Plcg1+/+, n = 6; Plcg1−/−, n = 6). (I) Peripheral blood chimerism of secondary recipient mice (Plcg1+/+, n = 10; Plcg1−/−, n = 11); shown are 2 independent cohorts. (J) Colony count of BM cells derived from healthy donors. Genetic inactivation of PLCG1 by shRNA compared with nontargeting control (shSCR). n = 5, in duplicate. (K) Short-term stress analysis after serial 5-fluorouracil (5-FU) injections; Kaplan-Meier survival curve of Plcg1+/+ (n = 9) and Plcg1−/− (n = 7) mice injected intravenously (i.v.) with 150 mg/kg 5-FU (arrows) every 7 days. (L) Long-term stress analysis by serial 5-FU injections (2 monthly injections IV). Kinetics of hematopoietic recovery as measured by peripheral white blood count of Plcg1−/− (n = 8) and Plcg1+/+ (n = 5) mice during the 2 monthly 5-FU injection schedule. (M) Survival rates of Plcg1−/− (n = 8) and Plcg1+/+ (n = 5) mice during long-term 5-FU treatment.

PLCG1 is dispensable for normal HSC function. (A) Experimental protocol for investigation of steady-state hematopoiesis. (B) White blood count (WBC), hemoglobin (HGB), and platelets (PLT) following genetic inactivation of Plcg1 (Plcg1−/−, n = 6) for 16 weeks of steady-state hematopoiesis, compared with Plcg1+/+ controls (n = 14). (C) Immunophenotypic quantification of mature myeloid (Gr-1 Mac-1; F4/80), B-lymphoid (B220; CD19), and T-lymphoid (CD3) bone marrow cells (Plcg1+/+, n = 10; Plcg1−/−, n = 6). (D) Immunophenotypic quantification of stem and progenitor cell abundance, specifically of hematopoietic stem cells (HSC: CD150+ CD48− L−S+K+) and multipotent progenitors (MPP: CD150 low, CD48+ L−S+K+) (Plcg1+/+, n = 10; Plcg1−/−, n = 6). (E) Protocol for assessing impact of Plcg1 loss on LT-HSC function by serial transplantation. (F) Peripheral blood chimerism of primary recipient mice (Plcg1+/+, n = 6; Plcg1−/−, n = 11); shown are 2 independent cohorts. (G) Immunophenotypic quantification of mature myeloid (Gr-1), B-lymphoid (B220; CD19), and T-lymphoid (CD3) bone marrow cells (Plcg1+/+, n = 6; Plcg1−/−, n = 6) from primary recipients. (H) Immunophenotypic quantification of stem and progenitor cell abundance, specifically of hematopoietic stem cells (HSC: CD150+ CD48− L−S+K+) and multipotent progenitors (MPP: CD150 low, CD48+ L−S+K+) (Plcg1+/+, n = 6; Plcg1−/−, n = 6). (I) Peripheral blood chimerism of secondary recipient mice (Plcg1+/+, n = 10; Plcg1−/−, n = 11); shown are 2 independent cohorts. (J) Colony count of BM cells derived from healthy donors. Genetic inactivation of PLCG1 by shRNA compared with nontargeting control (shSCR). n = 5, in duplicate. (K) Short-term stress analysis after serial 5-fluorouracil (5-FU) injections; Kaplan-Meier survival curve of Plcg1+/+ (n = 9) and Plcg1−/− (n = 7) mice injected intravenously (i.v.) with 150 mg/kg 5-FU (arrows) every 7 days. (L) Long-term stress analysis by serial 5-FU injections (2 monthly injections IV). Kinetics of hematopoietic recovery as measured by peripheral white blood count of Plcg1−/− (n = 8) and Plcg1+/+ (n = 5) mice during the 2 monthly 5-FU injection schedule. (M) Survival rates of Plcg1−/− (n = 8) and Plcg1+/+ (n = 5) mice during long-term 5-FU treatment.

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