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RNA interference. The target sequences for siRNA against the human NIBAN gene was 5′-GCATTTCCCAGACCCTCTTTT-3′ (sense strand). The random control sequence was 5′-GCTGCAATCGATTGATAGC-3′ (sense strand). HeLa PF-05212384 (PKI-587) were transfected with individual siRNAs (25 nM final) and Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
Results
Induction of Niban gene expression by tunicamycin
We examined Niban/NIBAN mRNA and protein expression patterns under various conditions using HeLa cells and renal tumor (RT) cell lines from model animals (see Supplementary Materials and methods). We found that TM, a potent endoplasmic reticulum (ER) stress inducer, increased the levels of Niban/NIBAN mRNA and protein, although basal levers and relative increases differed between cell lines ( Fig. 1A and B).
Fig. 1.
Increased levels of Niban/NIBAN following tunicamycin treatment. (A) Northern blot analysis. NR32, ERC33, and MKOC1-277 (MKOC) RT cells and HeLa cells were cultured either with 2 μg/ml tunicamycin (TM +) or vehicle only (?) for 20 h. RNAs were analyzed for the 6.5 kb Niban/NIBAN mRNA (Niban). Control 28S ribosome RNAs are shown below. (B) Western blot analysis. Cells were cultured as in (A) and analyzed with Niban antibodies (upper panel). The lower panel shows β-actin. (C) RT-PCR analysis of mouse organs. After 24 h TM treatment, Niban cDNA was amplified by RT-PCR (upper panel). Lanes, (+); TM-treated mouse, (?); vehicle-treated mouse. β-Actin cDNA was used as a control (lower panel). Results were confirmed with two-independent experiments.