Figure 4.

2B1 cells fail to accumulate rpL13a snoRNAs in the cytoplasm. (a) CHO and 2B1 cells were treated with 500 μM palm for the indicated times. Cytoplasmic RNA was isolated by sequential detergent extraction, reverse transcribed, and analyzed by qRT-PCR for rpL13a snoRNAs relative to β-actin. Graph (left) shows rpl13a snoRNA expression, and blots (right) show representative western analysis of fractions for hsp90 and histone H3 in cytosolic and nuclear fractions at 8 h. (b) CHO, 2B1 cells and 2B1 cells stably transfected with EV or plasmids encoding WT or CI murine RNASET2 (as in Figure 3) were treated with 500 μM palm for 10 h. Cytoplasmic RNA was isolated by sequential detergent extraction, reverse transcribed, and analyzed by qRT-PCR for rpL13a snoRNAs relative to actin (c–e). CHO (open bars) and 2B1 (filled bars) cells were untreated (−) or treated with palmitate for 9 h (+) and then fractionated by sequential detergent extraction. RNA from nuclear fractions was reverse transcribed and quantified by qPCR for pre-mRNA (c), intron lariats (d), and nuclear snoRNAs (e), each relative to actin. Graphs report mean values+S.E. for n=3 independent experiments. *P<0.05 for treated versus untreated; **P<0.05 for 2B1 versus CHO