Fig. 1: TET2 downregulation promotes MIF production.
From: Macrophage migration inhibitory factor is overproduced through EGR1 in TET2low resting monocytes
a, b Cytokine profile arrays of supernatant collected from cord blood CD34+ cells infected with SCR- and TET2-shRNA-GFP lentiviruses, sorted on GFP expression, and induced to differentiate with stem cell factor (SCF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT3L) and granulocyte-colony stimulating factor (G-CSF). a Representative cytokine array with supernatants collected at day 10 of differentiation. The rectangle points to MIF detection. b Quantification of MIF signals, normalized to positive controls. Data are mean +/− SEM of three independent experiments. Paired t test: *P < 0.05. c MIF concentrations determined by ELISA in the supernatant of cells induced to differentiate for indicated time. Data are mean +/− SEM of indicated independent experiments (day 5: n = 6; day 7: n = 5; day 8: n = 3; day 10: n = 7). Paired t test: *P < 0.05; ***P < 0.001. d RT-qPCR analysis of MIF mRNA expression in four TET2-depleted (TET2 shRNA, gray bars) and control (SCR shRNA, black bars) human leukemic cell lines. Data are mean +/− SEM of three biological replicates. Unpaired t test: *P < 0.05; ***P < 0.001; ****P < 0.0001. e Immunoblot of SCR or TET2 shRNA infected leukemic cell lines sorted on GFP expression. Lower panels, quantification after actin normalization using Image J software. f MIF concentrations determined by ELISA in the supernatants of kasumi-1 (n = 5), M07e (n = 5), UT-7 (n = 4) and TF-1 (n = 5) cells transduced 24 h before with SCR (black squares) or TET2 (gray squares) shRNA. Data are mean +/− SEM of indicated biological replicates. Unpaired-t test: *P < 0.05; **P < 0.01. g MIF concentrations determined by ELISA in the plasma of two Tet2-deficient models (1–3 months): K427 knock-out model (wt/wt or Tet2+/+ n = 4, wt/exc or Tet2+/− n = 5 and exc/exc or Tet2−/− n = 5); ANO knock-down model (wt/wt or Tet2+/+ n = 5, wt/LacZ or Tet2+/− n = 5 and LacZ/LacZ or Tet2−/− n = 6). Data are mean +/− SEM of indicated biological replicates. Dunnett’s multiple comparison tests using wt/wt as control: *P < 0.05, **P < 0.01, ***P < 0.001.
