Figure 5.
Inhibition of ISG15 impedes APL cell differentiation. NB4 cells expressing nontargeting shRNA (SHC) or shRNA targeting ISG15 (shISG15_319 and shISG15_352) were seeded at 0.2 × 105 cells per mL and treated for 72 h with 1 μm ATRA. (A) Functional knockdown efficiency was tested by measuring protein levels of both free and conjugated ISG15 in whole‐cell lysates by immunoblot at 72 h. β‐actin was used as a loading control. (B) Total RNA was extracted, and differentiation was assessed by measuring GCSFR mRNA expression by qPCR. Values are given as n‐fold induction compared with untreated cells and normalized to housekeeping gene HMBS (n = 3) (t‐test ****P ≤ 0.0001, ***P ≤ 0.001). (C) Surface CD11b protein expression on live cells was measured by flow cytometry as a second assay of differentiation. MFIs are shown at 72 h (n = 3) (t‐test *P ≤ 0.05). (D) Morphologic appearance of treated cells at 72 h. Neutrophil differentiation evidenced by increased cytoplasmic volume and nuclear lobulation indicated with arrows. (E) Neutrophil function was tested using nitro blue tetrazolium at 72 h. (i) Differentiated cells reduce nitro blue tetrazolium to blue color. (ii) nitro blue tetrazolium‐positive cells were counted in triplicate and presented as mean ± S.E.M (t‐test **P ≤ 0.01, *P ≤ 0.05) (magnification 400×).