Figure 4.
An HDAC 1 and 2-specific inhibitor, romidepsin, synergizes with 7C6 to increase the surface MICA/B expression levels. (A) Romidepsin increases MICB mRNA expression. NB4 cells were treated for 24 hours with 10 nmol/L of the indicated inhibitors or equal volume of dimethyl dioxide. Subsequently, mRNA was extracted, cDNA was synthetized, and PCR was performed with primers specific for the indicated genes plus GAPDH. Analyses were performed via electrophoresis and ImageJ software with GAPDH normalization. (B-C) Romidepsin increases MICB expression for stabilization of the surface protein by 7C6. NB4 cells were treated for 24 hours with 20 μg/mL of the indicated antibodies plus the indicated concentrations of romidepsin. Subsequently, soluble MICA/B shed into culture supernatants were analyzed by sandwich ELISA kits specific for either MICA or MICB. Surface MICA/B and cellular viability were analyzed by flow cytometry upon labeling of cells with 6D4 and Zombie Yellow, respectively. (D) Romidepsin + 7C6 treatment of leukemia cells enhances phagocytosis. NB4 cells were treated for 24 hours with romidepsin or equal volume of phosphate-buffered saline + 20 μg/mL of the indicated antibodies, and subsequently labeled with CFSE. Monocyte-derived macrophages from healthy donors were cocultured for 1 hour with the treated NB4 cells, and phagocytosis was analyzed by flow cytometry via identification of CD14+ CFSE+ macrophages. (E) Romidepsin and 7C6 increase the surface MICA/B expression levels in iPSC-derived AML blasts and LSC. The indicated cells were treated for 24 hours with the indicated concentrations of romidepsin plus 20 μg/mL of the indicated antibodies, and surface MICA/B was analyzed by flow cytometry. (F) Effects of romidepsin and 7C6 in CD45+ CD33+ CD34+ primary cells from de novo AML patients. Peripheral blood mononuclear cells from patients with AML were treated for 24 hours with the indicated doses of romidepsin plus 20 μg/mL of the indicated antibodies. Subsequently, the surface MICA/B levels were analyzed by flow cytometry with antibody panel (CD45, CD33, CD34, and MICA/B) plus Zombie Yellow. Romidepsin and 7C6 increase the surface MICA/B expression levels in AML patient-derived cells. Data are representative of (A-B,D-E) 3 independent experiments, and are (B,D-F) mean ± SD of triplicates, except for panel E LSC 0 nmol/L romidepsin isotype that are duplicates. The P values were calculated with 2-way ANOVA. ***P < .001 (calculated with Bonferroni’s post hoc test).

An HDAC 1 and 2-specific inhibitor, romidepsin, synergizes with 7C6 to increase the surface MICA/B expression levels. (A) Romidepsin increases MICB mRNA expression. NB4 cells were treated for 24 hours with 10 nmol/L of the indicated inhibitors or equal volume of dimethyl dioxide. Subsequently, mRNA was extracted, cDNA was synthetized, and PCR was performed with primers specific for the indicated genes plus GAPDH. Analyses were performed via electrophoresis and ImageJ software with GAPDH normalization. (B-C) Romidepsin increases MICB expression for stabilization of the surface protein by 7C6. NB4 cells were treated for 24 hours with 20 μg/mL of the indicated antibodies plus the indicated concentrations of romidepsin. Subsequently, soluble MICA/B shed into culture supernatants were analyzed by sandwich ELISA kits specific for either MICA or MICB. Surface MICA/B and cellular viability were analyzed by flow cytometry upon labeling of cells with 6D4 and Zombie Yellow, respectively. (D) Romidepsin + 7C6 treatment of leukemia cells enhances phagocytosis. NB4 cells were treated for 24 hours with romidepsin or equal volume of phosphate-buffered saline + 20 μg/mL of the indicated antibodies, and subsequently labeled with CFSE. Monocyte-derived macrophages from healthy donors were cocultured for 1 hour with the treated NB4 cells, and phagocytosis was analyzed by flow cytometry via identification of CD14+ CFSE+ macrophages. (E) Romidepsin and 7C6 increase the surface MICA/B expression levels in iPSC-derived AML blasts and LSC. The indicated cells were treated for 24 hours with the indicated concentrations of romidepsin plus 20 μg/mL of the indicated antibodies, and surface MICA/B was analyzed by flow cytometry. (F) Effects of romidepsin and 7C6 in CD45+ CD33+ CD34+ primary cells from de novo AML patients. Peripheral blood mononuclear cells from patients with AML were treated for 24 hours with the indicated doses of romidepsin plus 20 μg/mL of the indicated antibodies. Subsequently, the surface MICA/B levels were analyzed by flow cytometry with antibody panel (CD45, CD33, CD34, and MICA/B) plus Zombie Yellow. Romidepsin and 7C6 increase the surface MICA/B expression levels in AML patient-derived cells. Data are representative of (A-B,D-E) 3 independent experiments, and are (B,D-F) mean ± SD of triplicates, except for panel E LSC 0 nmol/L romidepsin isotype that are duplicates. The P values were calculated with 2-way ANOVA. ***P < .001 (calculated with Bonferroni’s post hoc test).

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