Induction of neutrophil secondary granule protein mRNAs in stably transformed NIH 3T3 cell lines expressing inducible human C/EBPε32 and rat C/EBPα.
(A) Western blot analysis of NIH 3T3 cell lines stably transformed with either empty (pMT), C/EBPε32, or C/EBPα containing zinc-inducible expression vector. Expression of C/EBPε32and C/EBPα was determined for cells incubated in the absence (−) or presence (+) of 0.1 mM ZnSO4 for 24 hours (lanes 1-6). COS-1 cells transfected with the same expression vectors and incubated with 0.1 mM ZnSO4 for 24 hours were included as negative and positive controls (lanes 7-9). (B) Northern blot analysis of total RNA prepared from the stable NIH 3T3 cell lines incubated in the absence (−) or presence (+) of 0.1 mM ZnSO4 for 48 hours. The blot was hybridized sequentially with probes for C/EBPε and C/EBPα, Β9, and β-actin. (C) Stable NIH 3T3 cell lines were incubated in the absence (−) or presence (+) of 0.1 mM ZnSO4 for 48 hours. Total RNA was harvested and subjected to RT-PCR analysis for the secondary granule genes B9 (33 cycles), MCLP (35 cycles), NGAL (33 cycles), NC (33 cycles), and the control gene GAPDH (25 cycles). Total RNA from undifferentiated 32Dcl3 cells was included as a positive control, but GAPDH expression was not determined (lane 7).
