Figure 4.
FOXOs induce HDAC8 transcription upon FLT3 inhibitor treatment. MV4-11 (A) and MOLM-13 (B) cells were treated with different inhibitors individually or in combination, as indicated, for 24 hours. Expression of the indicated proteins was analyzed with western blot. MV4-11 (C) and MOLM-13 (D) cells were transduced with vectors expressing control shRNA (shCtrl) or FOXO1 shRNA plus FOXO3 shRNA (shF1/F3). Doxycycline was added to induce shRNA expression for 48 hours. Then cells were treated with vehicle (dimethyl sulfoxide) or AC220 (5 nM) for another 24 hours. HDAC8 transcription was analyzed with quantitative real-time polymerase chain reaction (PCR). ns, P > .05, *P < .05, *** P < .001, ****P < .0001. MV4-11 (E) and MOLM-13 (F) cells, transduced with empty vectors (Vector) or FOXO1 plus FOXO3 expressing vectors (F1/F3 OE), were treated with AC220 (5 nM) for 24 hours. HDAC8 transcription was analyzed with quantitative real-time PCR. *P < .05, **P < .01, ***P < .001. Chromatin immunoprecipitation–quantitative PCR analysis of FOXO1 and FOXO3 binding to HDAC8 promoter in MV4-11 (G) and MOLM-13 (H) cells treated with vehicle or AC220 (5 nM). **P < .01, ***P < .001. MV4-11 (I) and MOLM-13 (J) cells, transduced with shCtrl or shF1/F3, were treated with vehicle or AC220 (5 nM) for 24 hours and subjected to western blot to detect the indicated proteins. shCtrl or shF1/F3 transduced MV4-11 (K) and MOLM-13 (L) cells (NC) were transduced with vectors expressing HDAC8 (HDAC8 OE) or p53 shRNA (p53 KD). Doxycycline was added to induce shRNA expression for 48 hours and then cells were treated with vehicle (dimethyl sulfoxide) or AC220 (5 nM) for another 24 hours. The apoptosis of cells was analyzed with annexin V/propidium iodide labeling. **P < .01, ***P < .001, ****P < .0001 vs vehicle;  ^P < .05,  ^^P < .01,  ^^^P < .001 vs NC. Data are mean ± standard error of the mean. ns, not significant (P > .05). KD, knockdown; NC, negative control; OE, overexpression.

FOXOs induce HDAC8 transcription upon FLT3 inhibitor treatment. MV4-11 (A) and MOLM-13 (B) cells were treated with different inhibitors individually or in combination, as indicated, for 24 hours. Expression of the indicated proteins was analyzed with western blot. MV4-11 (C) and MOLM-13 (D) cells were transduced with vectors expressing control shRNA (shCtrl) or FOXO1 shRNA plus FOXO3 shRNA (shF1/F3). Doxycycline was added to induce shRNA expression for 48 hours. Then cells were treated with vehicle (dimethyl sulfoxide) or AC220 (5 nM) for another 24 hours. HDAC8 transcription was analyzed with quantitative real-time polymerase chain reaction (PCR). ns, P > .05, *P < .05, *** P < .001, ****P < .0001. MV4-11 (E) and MOLM-13 (F) cells, transduced with empty vectors (Vector) or FOXO1 plus FOXO3 expressing vectors (F1/F3 OE), were treated with AC220 (5 nM) for 24 hours. HDAC8 transcription was analyzed with quantitative real-time PCR. *P < .05, **P < .01, ***P < .001. Chromatin immunoprecipitation–quantitative PCR analysis of FOXO1 and FOXO3 binding to HDAC8 promoter in MV4-11 (G) and MOLM-13 (H) cells treated with vehicle or AC220 (5 nM). **P < .01, ***P < .001. MV4-11 (I) and MOLM-13 (J) cells, transduced with shCtrl or shF1/F3, were treated with vehicle or AC220 (5 nM) for 24 hours and subjected to western blot to detect the indicated proteins. shCtrl or shF1/F3 transduced MV4-11 (K) and MOLM-13 (L) cells (NC) were transduced with vectors expressing HDAC8 (HDAC8 OE) or p53 shRNA (p53 KD). Doxycycline was added to induce shRNA expression for 48 hours and then cells were treated with vehicle (dimethyl sulfoxide) or AC220 (5 nM) for another 24 hours. The apoptosis of cells was analyzed with annexin V/propidium iodide labeling. **P < .01, ***P < .001, ****P < .0001 vs vehicle;  ^P < .05,  ^^P < .01,  ^^^P < .001 vs NC. Data are mean ± standard error of the mean. ns, not significant (P > .05). KD, knockdown; NC, negative control; OE, overexpression.

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