Figure 5
Figure 5. Panobinostat treatment of A/E9a;NrasG12D leukemic cells triggers differentiation. (A) Cell cycle analysis of A/E9a;NrasG12D leukemic cells treated in vitro with vehicle or 16 nM panobinostat for the indicated time. Percentage of cells in S phase (EdU positive) was determined by flow cytometry. Mean values of 2 independent experiments are shown; error bars represent standard deviation (SD). (B) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with vehicle (D) or 16 nM panobinostat (P) for the indicated time using antibodies to p16INK4A, p21WAF1/CIP1, and phosphorylated RB. β-actin served as loading control. The results shown are representative of 3 independent experiments. (C) Flow cytometry analysis of c-Kit expression in A/E9a;NrasG12D leukemic cells treated in vitro with 16 nM panobinostat for the indicated time. Mean values of 3 independent experiments are shown; error bars represent SD. (D) Quantitative real-time PCR of relative mRNA levels of target genes in A/E9a;NrasG12D leukemic cells treated in vitro with 16 nM panobinostat for 24 hours. Results were normalized to HPRT mRNA. Mean value of 3 to 6 individual experiments is shown, and error bars represent SD. (E) Quantitative real-time PCR of relative mRNA levels of target genes in GFP-positive splenocytes isolated from A/E9a;NrasG12D leukemia recipient mice 72 hours after initiation of treatment with panobinostat (25 mg/kg) or vehicle (D5W). Results were normalized to HPRT mRNA. Mean value of 3 to 5 individual samples is shown, and error bars represent standard error of the mean. (F-G) Flow cytometry analysis of c-Kit (F) and Mac-1 (G) expression in GFP-positive bone marrow cells isolated from A/E9a;NrasG12D leukemia recipient mice treated with panobinostat (25 mg/kg) or vehicle (D5W) for 3 days. Data are combined from 2 individual experiments. Each data point represents an individual mouse, and horizontal bars represent mean value. (F) *P = .0248; ***P = .0009. (G) *P = .0292. (H) Light microscopy of May-Grunwald/Giemsa–stained bone marrow cells isolated from A/E9a;NrasG12D leukemia recipient mice cells treated with panobinostat (25 mg/kg) or vehicle (D5W, 250 µL) for 5 days. Imaging was performed with a ×60 objective. Representative images of 5 biological replicates are shown (bars represent 10 µm). GFP-positive cells isolated from 5-day vehicle-treated mice (left panel) demonstrate immature blast morphology, including a fine rim of agranular basophilic cytoplasm with a round to oval nucleus containing “open chromatin” and 1 or more prominent nucleoli (arrowheads). In contrast, GFP-positive cells from 5-day panobinostat-treated mice (right panel) show features of maturation including a reduction in the nuclear:cytoplasmic ratio, condensation of nuclear chromatin, and infrequent nucleolation. Frequent coarse azurophilic cytoplasmic granules (arrowheads) indicate myeloid differentiation.

Panobinostat treatment of A/E9a;NrasG12Dleukemic cells triggers differentiation. (A) Cell cycle analysis of A/E9a;NrasG12D leukemic cells treated in vitro with vehicle or 16 nM panobinostat for the indicated time. Percentage of cells in S phase (EdU positive) was determined by flow cytometry. Mean values of 2 independent experiments are shown; error bars represent standard deviation (SD). (B) Western blot analysis of whole-cell lysates prepared from A/E9a;NrasG12D leukemic cells treated in vitro with vehicle (D) or 16 nM panobinostat (P) for the indicated time using antibodies to p16INK4A, p21WAF1/CIP1, and phosphorylated RB. β-actin served as loading control. The results shown are representative of 3 independent experiments. (C) Flow cytometry analysis of c-Kit expression in A/E9a;NrasG12D leukemic cells treated in vitro with 16 nM panobinostat for the indicated time. Mean values of 3 independent experiments are shown; error bars represent SD. (D) Quantitative real-time PCR of relative mRNA levels of target genes in A/E9a;NrasG12D leukemic cells treated in vitro with 16 nM panobinostat for 24 hours. Results were normalized to HPRT mRNA. Mean value of 3 to 6 individual experiments is shown, and error bars represent SD. (E) Quantitative real-time PCR of relative mRNA levels of target genes in GFP-positive splenocytes isolated from A/E9a;NrasG12D leukemia recipient mice 72 hours after initiation of treatment with panobinostat (25 mg/kg) or vehicle (D5W). Results were normalized to HPRT mRNA. Mean value of 3 to 5 individual samples is shown, and error bars represent standard error of the mean. (F-G) Flow cytometry analysis of c-Kit (F) and Mac-1 (G) expression in GFP-positive bone marrow cells isolated from A/E9a;NrasG12D leukemia recipient mice treated with panobinostat (25 mg/kg) or vehicle (D5W) for 3 days. Data are combined from 2 individual experiments. Each data point represents an individual mouse, and horizontal bars represent mean value. (F) *P = .0248; ***P = .0009. (G) *P = .0292. (H) Light microscopy of May-Grunwald/Giemsa–stained bone marrow cells isolated from A/E9a;NrasG12D leukemia recipient mice cells treated with panobinostat (25 mg/kg) or vehicle (D5W, 250 µL) for 5 days. Imaging was performed with a ×60 objective. Representative images of 5 biological replicates are shown (bars represent 10 µm). GFP-positive cells isolated from 5-day vehicle-treated mice (left panel) demonstrate immature blast morphology, including a fine rim of agranular basophilic cytoplasm with a round to oval nucleus containing “open chromatin” and 1 or more prominent nucleoli (arrowheads). In contrast, GFP-positive cells from 5-day panobinostat-treated mice (right panel) show features of maturation including a reduction in the nuclear:cytoplasmic ratio, condensation of nuclear chromatin, and infrequent nucleolation. Frequent coarse azurophilic cytoplasmic granules (arrowheads) indicate myeloid differentiation.

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