Figure 3

Knocking-down BIRC6 attenuates neutrophil differentiation of APL cells. (A) NB4 BIRC6 knock-down cells (shBIRC6) were generated by lentiviral transduction of shRNAs targeting the BIRC6 mRNA. BIRC6 knockdown efficiency compared to control transduced (SHC002) NB4 cells was determined by qPCR analysis. Values are shown as n-fold regulation compared to control transduced cells. (B,C) NB4 shBIRC6 and SHC002 were treated with 1 μM ATRA for 4 and 6 days, respectively. Neutrophil differentiation was determined by G-CSFR mRNA and CD11b surface expression. The raw Ct values of G-CSFR were normalized to HMBS and to the Ct values of the respective control transduced cells under ATRA treatment. CD11b expression is shown as mean fluorescence intensity (MFI). (D,E) Cell death in BIRC6 knock-down cells at day 4 and 6 of ATRA treatment was assessed by AnnexinV staining and Trypanblue exclusion assays. Data are given as percentage or total number of death cells, respectively. (F) Total cell counts were determined using a hemocytometer. There was no difference in total cell number at day 4 or 6 of ATRA treatment between shBIRC6 or control NB4 cells. Mann–Whitney U test **p < 0.01, ***p < 0.001, ns not significant.