Proceedings of the National Academy of Sciences of the United States of America 20130318 14
The unfolded protein response (UPR) is a cellular response highly conserved in eukaryotes to obviate accumulation of misfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) catalyzes the cytoplasmic splicing of mRNA encoding bZIP transcription factors to activate the UPR signaling pathway. Arabidopsis IRE1 was recently shown to be involved in the cytoplasmic splicing of bZIP60 mRNA. In the present study, we demonstrated that an Arabidopsis mutant with defects in ...[more]
Project description:We measured miRNA turnover rates in 8 mammalian cell types post transcription inhibitors ActinomycinD (ActD) or 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) treatment with miRNA expression profiling, generating 218 expression profiles on 528 endogenous miRNAs. Cells were plated in six-well plates at around 200,000 cells/well. A media change was performed one day after plating. ActD and DRB treatment were started after confluency (around two days after plating) for 0, 2, 4, 6, 12, and 24 hours post treatment with biological duplicate (ActD treatment) or biological triplicate (DRB treatment). The concentration of ActD and DRB for different cell lines were titrated to achieve rapid c-myc down-regulation after 2 hours of ActD and DRB treatment. After treatment at indicated time points, cells were washed with PBS once and lysed in TriZol reagent immediately (Invitrogen). RNA extraction was performed following the manufacturer’s protocol. miRNA profiling was performed using a Luminex bead-based method.
Project description:The goal of this experiment was to assess whether transcription happens during mitosis. For this, we isolated mitotic HeLa cells and treated them with a transcription inhibitor, ActD.
Project description:Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.
Project description:HDMYZ cells were treated with 4ug/ml ActD for 0, 1, 4 and 12 hours. Small RNAs of 15-40 bases were gel-purified from 5 ug total RNA, and subjected to multiplex Illumina small RNA library preparation. Small RNA libraries were sequenced on a HiSeq2000 (Illumina). Measure miRNA expression level at 0, 1, 4, 12 hours post ActinomycinD treatment to examine miRNA turnover pattern.
Project description:We investigate the contribution of IRE1 signaling to the modulation of U87 glioma cells transcriptome upon various stresses. To this end, IRE1 control and IRE1 dominant negative expressing U87 glioma cells were subjected to environmental or chemical challenges and their transcriptome monitored using Affymetrix microarrays. Stress-induced transcriptome modulation in function of IRE1 proficiency/deficiency
Project description:7-days-old Arabidopsis seedlings of wildtype (Col-0) were treated with 1 μM IAA for 15 minutes or 3 hours and gene expression of whole plant was analyzed using Affymetrix Gene 1.1 ST Array strips. Arabidopsis seedling (Col-0) 25 plants were grown in each plate (1/2 MS medium) under continuous light at 22 °C for 6 or 7 days. Ten seedlings were cultured in 50 ml plastic tube containing liquid medium (1/2 MS medium), and incubated with shaking. After 24 hours, samples were treated with 0.1% DMSO for 15 minutes or 3 hours (Mock treatments) and with 1 μM indole-3-acetic acid dissolved in DMSO for 15 minutes or 3 hours (IAA tretments). The plants were wiped on papertowels and sampled. Immediately after sampling of the plants, these plants were soaked in liquid nitrogen and stored at -80 °C. The experiment was repeated 3 times in 3 different days. Total RNA was extracted from the frozen samples using RNeasy Plant Mini Kit (Qiagen) following the protocol supplied by manufacturer. The quality of RNA was checked by 2100 Bioanalyzer system (Agilent) and 100 ng RNA of each experiment was applied to microarray analysis. The microarray analysis was carried out using Affymetrix gene 1.1 ST array strips and GeneAtlas (Affymetrix) following the protocol supplied manufacturer.
Project description:Transcriptional profiling of murine hepatocyte gene expression following exposure to prolactin or actinomycin D Prolactin or actinomycin D-treated versus serum-free control cells: AML12 and Hepa 1-6. Biological replicates: 2 per cell line per treatment protocol.