Fig. 6: O-GlcNAc modification is a potential contributor for TRAF6-mediated metabolic reprogramming of leukemia.

Immunoblot analysis of OGT in HEL (A) and TF-1 (B) transduced with inducible shTRAF6 expressing either control vector or cDNA of OGT, cultured with or without DOX (1 μg/mL) for 3 days (left panel). Viable cell growth of the cells was assayed by trypan blue exclusion (right panel). The normalized cell count, relative to untreated cells, was determined 72 h post-seeding of an equal number of cells. Data are presented as the means ± SD from technical triplicates. Results are representative of two independent assays. C OCR in HEL cells transduced with inducible shTRAF6, expressing control vector or cDNA of OGT, untreated or treated with DOX for 3 days. Cells were sequentially treated with oligomycin. FCCP, and rotenone/antimycin A at the indicted time points. Data are shown as the means ± SD for technical replicate analyses (n = 6). Results are representative of two independent assays. D Basal respiration, maximal respiration, ATP production and spare respiratory capacities of HEL cells calculated from the data of (C). The data are shown as the means ± SD (n = 6). E Schematic of O-GlcNacylation. F Immunoblotting of HEL cells transduced with the inducible shTRAF6, treated with or without DOX (1 μg/mL) for 3 days. G Immunoblotting of HEL cells transduced with the inducible shTRAF6, untreated with DOX, treated with DOX, and treated with DOX and 100 nM of MK8719 (OGA inhibitor). H One hundred thousand HEL cells transduced with inducible shTRAF6 were cultured with 1 μM of MK8719 for 7 days. Viable cell growth of the cells was assayed by trypan blue exclusion. Data are presented as the means ± SD for technical triplicates. Results are representative of two independent assays. I OCR in HEL cells transduced with the inducible shTRAF6 untreated with DOX, treated with DOX (1 μg/mL), and treated with DOX (1 μg/mL) and 200 nM of MK8719 (OGA inhibitor). Cells were sequentially treated with oligomycin. FCCP, and rotenone/antimycin A at the indicted time points. Data are presented as the means ± SD from technical replicate analyses (n = 3–4). Results are representative of two independent assays. J Basal respiration, maximal respiration, ATP production and spare respiratory capacities of HEL cells transduced with the inducible shTRAF6 calculated from the data of (I). Data are shown as the means ± SD (n = 3–4). *P < 0.05; **<0.01; ***P < 0.001.