Cycloheximide and heat shock induce a significant increase in the level of co-expression of Alu and its downstream miRNA. A. Northern blotting of full length Alu transcripts after induction by heat shock or cycloheximide. Total RNAs extracted from HEK293 cells were separated by urea PAGE and hybridized with flAlu (full length Alu) or 5S rRNA probes. Northern blotting was performed using the Typhoon 9410 Phosphorlmager. The data are averages of at least three independent determinations. The error bars indicate standard deviations. *P < 0.001; cycloheximide or heat shock compared with the control group. B. Northern blotting was used to detect changes in Alu expression in HeLa cells after cycloheximide or heat shock induction. C. RT-PCR of expression of the host gene ATAD3B in HEK293 and HeLa cells after induction by heat shock and cycloheximide. Beta-actin was taken as a loading control. D. After cycloheximide and heat shock induction, the expression levels of Alu-related miRNAs in H293 cells were examined by real-time PCR. 5sRNA was taken as a loading control. The expression levels of miRNA were normalized to that of 5sRNA and the results in different cases were normalized to the expression levels in the control. The results are representative triplicate experiments and are given as mean and S.D. E. After cycloheximide and heat shock induction, real-time PCR was used to detect changes in Alu-related miRNAs in HeLa cells. F. Northern blotting showing inhibition of Pol-III or Pol-II activity by tagetitoxin. Let-7a was taken as both negative and loading control.
Gu et al. BMC Genomics 2009 10:563 doi:10.1186/1471-2164-10-563