siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments

Kaempferol induced the apoptosis via cell cycle arrest in human breast cancer MDA-MB-453 cells

The aim of present study was to investigate the effects of kaempferol on cellular proliferation and cell cycle arrest and explore the mechanism for these effects in human breast carcinoma MDA-MB-453 cells. Cells were treated with kaempferol at various concentrations (ranging from 1 to 200 µM) for 24 and 48 hrs. Kaempferol significantly inhibited cancer cell growth in cells exposed to 50 and 10 µM of kaempferol and incubated for 24 and 48 hrs, respectively. Exposure to kaempferol resulted in cell cycle arrest at the G2/M phase. Of the G2/M-phase related proteins, kaempferol down-regulated CDK1 and cyclin A and B in cells exposed to kaempferol. In addition, small DNA fragments at the sub-G0 phase were increased by up to 23.12 and 31.90% at 10 and 50 µM incubated for 24 and 48 hrs, respectively. The kaempferol-induced apoptosis was associated with the up-regulation of p53. In addition, the phosphorylation of p53 at the Ser-15 residue was observed with kaempferol. Kaempferol inhibits cell proliferation by disrupting the cell cycle, which is strongly associated with the induction of arrest at G2/M phase and may induce apoptosis via p53 phosphorylation in human breast carcinoma MDA-MB-453 cells

Observation of inter-subunit nuclear Overhauser effects in a dimeric protein Application to the Arc repressor

AbstractFor the structure determination of symmetric protein dimers it is necessary to distinguish between intra- and inter-subunit NOEs. A method is presented to measure selectively the inter-subunit NOEs using uniform 15N and 13C isotope labelling. This is accomplished by doubly filtered 2D NOE experiments on mixtures of native protein with isotope-labeled protein. The method has been applied to the Arc represser and allows the characterization of virtually all proton-proton NOEs in terms of their intra- or inter-subunit nature