CXXC5 attenuates Wnt signaling in leukemic cells. Cells overexpressing EGFP-tagged CXXC5 or stably infected with lentiviral CXXC5 shRNA were treated with 100 ng/mL recombinant Wnt3a. (A) Decreased upregulation of total β-catenin and active β-catenin 5 hours after Wnt3a treatment in CXXC5-overexpressing cells compared with vector controls (Western blot analysis). A representative figure is shown (transfection efficiency of 59% for K562 and 42% for KG1a by flow cytometry). (B) Decreased upregulation of Wnt target genes Axin2 and Survivin 10 hours after Wnt3a treatment in CXXC5-overexpressing cells compared with vector controls as determined by RT-qPCR. Mean values of 2 independent experiments with standard deviations are shown (K562 Axin2: 1.4-fold vs 3.1-fold; K562 Survivin: 0.9-fold vs 1.2-fold; KG1a Axin2: 0.7-fold vs 1.7-fold; KG1a Survivin: 0.6-fold vs 1.3-fold; both P ≤ .05 by the Mann-Whitney U test[*]). (C) Impaired Wnt activation as determined by Wnt reporter assay activity 5 hours after Wnt3a treatment in CXXC5-overexpressing cells compared with vector controls. Mean values of 2 independent experiments with standard deviations are shown (K562: 4.7-fold vs 19.9-fold; KG1a: 0.9-fold vs 2.8-fold; *P ≤ .05 and **P ≤ .01 by the Mann-Whitney U test). (D) Impaired cell proliferation after CXXC5 overexpression (i, iii) and increased proliferation after CXXC5 knockdown (ii, iv) as determined by cell count (trypan blue staining; iii-iv) and MTT assay (i-ii) 24, 48, and 72 hours after start of treatment (days 1-3). Wnt3a protein was replaced every 24 hours. Mean values of 2 independent experiments performed in quadruplicates with standard deviations are shown. Co, vector control; scr, scrambled shRNA control; shR, CXXC5 shRNA. P values are shown in the figure (**P ≤ .01 and ***P ≤ .001 by the Mann-Whitney U test).