Fig 1: PRDX2 blocks apoptosis of myeloid leukemia cells. (A) Silenced PRDX2 using two independent shRNA and scrambled (shLuc) K562 cells were treated with 300 nM ABT-263 or without drug for 72 hours, and cell proliferation was measured by trypan blue exclusion method. (B) Colony formation ability was assayed in Methocult medium after being stably silenced of PRDX2 in K562 cells. After 7 days, colonies were photographed and counted manually; original magnification: ×100. (C) PRDX2-silenced and scrambled K562 cells treated with 300 nM ABT-263 for 72 hours. Apoptosis cells were analyzed using Annexin V and PI staining by flow cytometric analysis. Representative flow cytometry analysis (left panel) and the % of Annexin V and PI-positive cells are shown (right panel). (D) Immunoblot data showing the PRDX2 silenced efficiency in K562 cells and protein expression of P-MYC and MYC after knock down of PRDX2 in K562 cells. β-Actin was used as a control for equal loading. (E) Proliferation of U937 cells transduced with WT- or R882H and knocked down of PRDX2 from transduced cells. (F) Apoptosis analysis of R882H-expressed U937 cells knocked down with shLuc or shPRDX2 and treatment of 300 nM ABT-263 for 72 hours. (G) Immunoblot data showing the PRDX2 silenced efficiency in U937 cells transduced with R882H and protein expression of MYC and CDK1. β-Actin was used as a control for equal loading. All studies were repeated at least once. The mean ± S.D. of at least two replicates was plotted. *P < .05; **P < .03; ***P < .01.
Fig 2: DNMT3A-R882 mutants augmented PRDX2 expression in myeloid leukemia cells. (A and B) Immunoblot showing overexpression of DNMT3A-mutant into U937 (A) and HL-60 (B) cells augmented protein expression of PRDX2 with increasing MYC and P-MYC expression. β-Actin was used as a control for equal loading. (C) Cytoplasmic localization of PRDX2 in U937 cells transduced with EV-, WT-, and mutant-DNMT3A; immunostained with anti-PRDX2 (red) and DAPI (blue) (original magnification: ×1000). All the images were taken with same contrast and exposure time. (D) Co-immunoprecipitation data showing FLAG-tagged WT- and mutant DNMT3A both interacted with PRDX2 in 293T cells. Cell extracts were incubated with DNAse prior to immunoprecipitation (Supplementary Fig. S1 for effectiveness of DNAse treatment).
Fig 3: The proposed mechanism of impairment of apoptosis by DNMT3A-R882C/H mutant in myeloid cells. DNMT3A-R882 mutation-induced hypomethylation increased PRDX2 expression which reduced ROS accumulation in cells decreasing apoptosis by increasing antioxidant capacity of cells.
Fig 4: DNMT3A-R882 mutants induce modifications of genomic methylation patterns and proposed mechanism of impairment of apoptosis in myeloid cells. (A) Distribution of genomic methylation patterns (β-value) in whole genome of U937 cells transduced with EV control, R882C, and R882H is shown. (B) Hypo- and hypermethylation probes counts obtained from U937 cells transduced with control and mutant DNMT3A shown in bar diagram. β-value <0.25 and >0.75 considered as hypomethylation and hypermethylation peaks, respectively. (C) The total hypermethylation and hypomethylation probes counted in each region defined by genomic structure shown in bar graph. (D) Methylation patterns in four regions defined by the distance from CpG islands, such as CpG islands, Shore, Shelf, and Open Sea region, are shown. (E) Validation of CpG methylation of PRDX2 gene by genomic bisulfite methylation sequencing showing PRDX2 gene was hypomethylated in DNMT3A mutant U937 cells. (F) Immunoblot data showing the PRDX2 expression induced by the treatment of 10 μM AZACITIDINE (Sigma) for 72 hours in U937 cells transduced with EV and DNMT3A-R882H/C. β-Actin was used as a control for equal loading. The number below the lanes indicates relative band intensity normalized to actin.
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