Figure 6
Figure 6. Treatment of Eµ-Myc lymphoma or APL cells with HDAC3-selective RGFP966 mimics HDAC3 depletion. Eµ-Myc lymphoma and APL cells were treated with low micromolar concentrations of RGFP966 (≤2 µM) in vitro and assessed for apoptosis and cell proliferation. (A) Assessment of apoptosis (Annexin V/PI) in Eµ-Myc cells (no. 107) after 48 hours of treatment with RGFP966 using flow cytometry (n = 3 biological replicates; *P < .05). Data were analyzed using a one-way ANOVA. (B) Proliferation of Eµ-Myc (no. 107, n = 3 biological replicates) and Eµ-Myc.Bcl-2 (n = 2 biological replicates) cells was assessed using CTV staining following RGFP966 treatment (≤1 µM, 48 hours). Cells were analyzed for cell division and measured as discrete peaks of decreasing CTV fluorescence (PacBlue+ cells) using flow cytometry. Red ovals highlight the CTV peaks that demonstrate anti-proliferative effects of RGFP966. (C) APL cells were treated with RGFP966 at the indicated concentrations and subjected to colony-forming assays whereby cells were plated in methylcellulose (1 × 104 cells), left for 7-10 days, counted, and replated. Serial replating of RGFP966-treated APL cells led to a dose-dependent reduction in colony forming potential. Data are presented as mean number of colonies counted after 7-10 days ± SEM from 3 biological replicates. (D) Histological assessment of APL cell maturity demonstrates that RGFP966 treatment increases the population of mature vs immature cells in vitro (×60 magnification; ×4 representative cytospins; (top left) dimethylsulfoxide (DMSO); (top right) 0.05 µM RGFP966; (bottom left) 0.1 µM RGFP966; (bottom right) 0.5 µM RGFP966). At least 300 cells were scanned for each case (n = 3 biological replicates). (E) Calculated percentage of mature and immature cells following treatment with 0.05 µM, 0.1 µM, or 0.5 µM RGFP966. In addition, APL cells treated with RGFP966 at indicated concentrations were immunophenotyped by flow cytometry. (F) An increased percentage of cells positive for Gr.1(Ly-6G) or Mac.1 (CD11b) confirms that low concentrations of RGFP966 triggers differentiation in APL cells similar to Hdac3 depletion (n = 2 biological replicates).

Treatment of Eµ-Myc lymphoma or APL cells with HDAC3-selective RGFP966 mimics HDAC3 depletion. Eµ-Myc lymphoma and APL cells were treated with low micromolar concentrations of RGFP966 (≤2 µM) in vitro and assessed for apoptosis and cell proliferation. (A) Assessment of apoptosis (Annexin V/PI) in Eµ-Myc cells (no. 107) after 48 hours of treatment with RGFP966 using flow cytometry (n = 3 biological replicates; *P < .05). Data were analyzed using a one-way ANOVA. (B) Proliferation of Eµ-Myc (no. 107, n = 3 biological replicates) and Eµ-Myc.Bcl-2 (n = 2 biological replicates) cells was assessed using CTV staining following RGFP966 treatment (≤1 µM, 48 hours). Cells were analyzed for cell division and measured as discrete peaks of decreasing CTV fluorescence (PacBlue+ cells) using flow cytometry. Red ovals highlight the CTV peaks that demonstrate anti-proliferative effects of RGFP966. (C) APL cells were treated with RGFP966 at the indicated concentrations and subjected to colony-forming assays whereby cells were plated in methylcellulose (1 × 104 cells), left for 7-10 days, counted, and replated. Serial replating of RGFP966-treated APL cells led to a dose-dependent reduction in colony forming potential. Data are presented as mean number of colonies counted after 7-10 days ± SEM from 3 biological replicates. (D) Histological assessment of APL cell maturity demonstrates that RGFP966 treatment increases the population of mature vs immature cells in vitro (×60 magnification; ×4 representative cytospins; (top left) dimethylsulfoxide (DMSO); (top right) 0.05 µM RGFP966; (bottom left) 0.1 µM RGFP966; (bottom right) 0.5 µM RGFP966). At least 300 cells were scanned for each case (n = 3 biological replicates). (E) Calculated percentage of mature and immature cells following treatment with 0.05 µM, 0.1 µM, or 0.5 µM RGFP966. In addition, APL cells treated with RGFP966 at indicated concentrations were immunophenotyped by flow cytometry. (F) An increased percentage of cells positive for Gr.1(Ly-6G) or Mac.1 (CD11b) confirms that low concentrations of RGFP966 triggers differentiation in APL cells similar to Hdac3 depletion (n = 2 biological replicates).

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