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Protein disulfide isomerase as a target in leukemia
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Figure 4.SK053 increases CEBPA levels in HL-60 cells and protein disulfide isomerase knock-down impairs the growth of acute myeloid leukemia cells and induces differenti- ation of HL-60 cells. (A) HL-60 cells were incubated for 2 or 5 days with 10 mM SK053. Subcellular fractions were isolated using a NE-PER kit. HDAC2 served as a loading control for the nuclear fraction. A representative result of a series of experiments is presented. (B) HL-60 cells were incubated for 1-120 h with 10 mM SK053, harvested, lysed and total cell lysates were immunoblotted for 30 kDa C/EBPa. a-tubulin levels served as a loading con- trol. A representative result of a series of experiments is presented. For (A) and (B) the con- trols are HL-60 cells incubated with dimethylsulfoxide at the same concentration as used for SK053-treated group, harvested on day 5. (C) Real-time quantitative polymerase chain reaction of HL-60 cells incubated for the indicated times with 10 mM SK053; results are presented as mean target-to-reference ratio ± SD of three experiments. (D) Western blot showing the efficacy of PDI or thioredoxin (TXN) knock-down 5 days after transduction with shRNA-encoding lentiviruses. PDI(a) and PDI(b) indicate two different shRNA; β-actin (βA) served as a loading control. (E) Growth of HL-60 cells transduced with non-targeting (NTC), TXN [TXN(a)]- or PDI [PDI(b)]-targeting shRNA lentiviral particles. Cells were counted daily in trypan blue from day 1 until day 8. *P<0.05 vs. NTC in a two-tailed Student t-test. For (C) and (D) the controls are non-modified HL-60 cells. (F) Western blot showing the efficacy of PDI knock-down in AML cell lines transduced with PDI-targeting shRNA [PDI(d)]-encod- ing lentiviruses. GAPDH served as a loading control. (G) Growth of HL-60, MOLM14 and KG1 cells transduced with non-targeting (NTC) or PDI-targeting [PDI(d)] shRNA lentiviral particles. Cells were counted daily in trypan blue from day 1 until day 9. *P<0.05 vs. NTC in a two-tailed Student t-test. (H) Mean percentage ± SD of AML cells modified with non- targeting (NTC) or PDI-targeting [PDI(d)] shRNA, expressing CD11b myeloid marker deter- mined in flow cytometry (n=4 experiments, except for KG1: n=2), ***P<0.0001 vs. NTC, two-tailed Student t-test; NS: not significant.
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