Skip to main content
. 2021 Jul 23;11(12):3198–3213. doi: 10.1158/2159-8290.CD-21-0177

Figure 4.

Figure 4. ActD activates a ROS/PML/TP53 senescence axis ex vivo. A, Quantification (left) and representative image (right) of PML NBs upon ActD exposure in MEFs stably expressing GFP-PML-III and transiently expressing NPM1 (WT) or NPM1c (C+). The results are expressed as the mean value ± SD (error bars) of n = 10 cells per condition. Unpaired t test; ***, P < 0.001. Scale bar, 10 μm. B, Ex vivo ActD treatment of primary AML blasts demonstrates PML NB restoration in NPM1c-AMLs. Representative images (left) and quantification (right). C, Histidine pulldown of disulfide-linked NPM1c–PML complex in transiently transfected 293T cells, before or after ActD exposure. D, Western blot analyses of AML3 and AML3PML−/− cells after ActD treatment. E, SA-β-gal staining of AML3 derivatives 7 days after 2 hours ActD pretreatment. F, Effect of ActD pretreatment on colony formation in the indicated AML cell lines. The results are expressed as the mean value of triplicate samples ± SD. Representative experiment of n = 2. Unpaired t test; ***, P < 0.001. G, Western blot analyses of NAC pretreated AML cells upon ActD exposure. H, Effect of NAC and ActD pretreatment on colony formation in the indicated AML cell lines. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test; ***, P < 0.001. Representative experiment of n = 2. I, Western blot analyses of ActD response of parental and mitochondria-depleted AML2 and AML3 cells. MT-CO1, Mitochondrial cytochrome c oxidase subunit 1; RXR, retinoid X receptor alpha.

ActD activates a ROS/PML/TP53 senescence axis ex vivo. A, Quantification (left) and representative image (right) of PML NBs upon ActD exposure in MEFs stably expressing GFP-PML-III and transiently expressing NPM1 (WT) or NPM1c (C+). The results are expressed as the mean value ± SD (error bars) of n = 10 cells per condition. Unpaired t test; ***, P < 0.001. Scale bar, 10 μm. B,Ex vivo ActD treatment of primary AML blasts demonstrates PML NB restoration in NPM1c-AMLs. Representative images (left) and quantification (right). C, Histidine pulldown of disulfide-linked NPM1c–PML complex in transiently transfected 293T cells, before or after ActD exposure. D, Western blot analyses of AML3 and AML3PML−/− cells after ActD treatment. E, SA-β-gal staining of AML3 derivatives 7 days after 2 hours ActD pretreatment. F, Effect of ActD pretreatment on colony formation in the indicated AML cell lines. The results are expressed as the mean value of triplicate samples ± SD. Representative experiment of n = 2. Unpaired t test; ***, P < 0.001. G, Western blot analyses of NAC pretreated AML cells upon ActD exposure. H, Effect of NAC and ActD pretreatment on colony formation in the indicated AML cell lines. The results are expressed as the mean value of triplicate samples ± SD. Unpaired t test; ***, P < 0.001. Representative experiment of n = 2. I, Western blot analyses of ActD response of parental and mitochondria-depleted AML2 and AML3 cells. MT-CO1, Mitochondrial cytochrome c oxidase subunit 1; RXR, retinoid X receptor alpha.