Mutation or knockdown of TET2 lead to decreased levels of 5-hmC in human hematopoietic cells. (A) 5-hmC content of granulocytes from healthy patients (controls) and wild-type (wt) or mutant (m) TET2 MPN patients was determined by analyzing serial 2-fold dilutions of granulocyte DNA on dot blots with an anti–5-hmC antibody. The membranes were stained by MB to allow quantification of spot intensities by ImageJ software. (B) The ratios of 5-hmC to MB spot intensities from 4 experiments (10 control samples, 14 wild-type TET2 MPN samples, and 9 mutant TET2 MPN samples) were calculated, and individual values were normalized to the average value of control DNA for each experiment. Normalized values are plotted as squares, triangles, diamonds, and circles, each symbol representing one experiment. Horizontal bars indicate median values. P values were obtained with the use of the 2-tailed Mann-Whitney test. (C) TET2 mRNA quantification in TF1, Kasumi-1, UKE1, and MO7e cell lines transduced by lentiviruses expressing GFP and shRNA designed against either TET2 (shRNA TET2) or a control scramble sequence (shRNA scramble). Error bars indicate SEM. (D) Western blot analysis of transduced cell lines with anti-TET2 and anti-HSC70 antibodies. EBV lymphoblastoid cell lines from a patient with no TET2 mutation (EBV TET2 pos) and a patient with a biallelic deletion of TET2 (EBV TET2 del) were used as positive and negative controls. (E) 5-hmC content was determined by HPLC-MSMS in transduced MO7E cells. Histograms show 5-hmC/5-mC ratios (mean of 3 experiments). Error bars indicate SEM. P value was obtained with an unpaired Student t test. (F) A total of 1-2 μg of DNA from transduced TF1, Kasumi-1, UKE1, and MO7e cells was spotted for 5-hmC dot blot assay. Membranes were stained with MB to control spotting.