Fig. 1
Stress granules and P-bodies are assembled after B-cell activation. a Quantitation by RT-qPCR of mRNA expression of PBs and SGs components in B cells after LPS activation. Data from at least two independent experiments are shown as mean + s.e.m. (Mann–Whitney test, ns non-significant, * P < 0.05, **P < 0.01, ***P < 0.001). b Immunoblot analysis of Dcp1a and Tia1 protein abundance in LPS- activated B cells. c Visualization of Dcp1a and Tia1 containing granules in ex vivo and LPS-activated B cells by confocal microscopy. Nuclei were stained using Dapi. The number of Dcp1a and Tia1- containing granules per cell is shown in the right panels. Quantitation was performed using Volocity Image software based on fluorescence accumulation within granules. A minimum of six confocal images from three independent experiments were analyzed. Data shown as mean + s.d. Statistical analysis of the number of objects in treated B cells compared to untreated B was performed using a Mann–Whitney test. (Scale bar = 10 μm). d Detection and feature distribution analysis of Tia1 binding to its mRNA targets in the cytoplasm of B cells treated with LPS for 48 h. Top panel, autoradiography showing Tia1:RNA complexes labelled with 32P-ATP after complete or partial RNA digestion with RNaseI. Red box indicates the RNA:protein complexes purified for cDNA library preparation and iCLIP analysis. Bottom panel, distribution analysis of iCLIP cDNA counts mapped to the indicated genomic features (3′UTR, 5′UTR, ORF, intergenomic, intron and ncRNA). Data from the two Tia1 iCLIP experiments performed using B-cell cytoplasmic extracts are shown. e Venn diagram showing the number of Tia1 mRNA targets identified in two iCLIP experiments. Only mRNA targets with at least one significantly enriched binding site in the 3′UTR were considered (transcripts collapsed by gene id, peak enrichment analysis, FDR < 0.05). f Gene ontology (GOrilla and REViGO) analysis of Tia1 mRNA targets in activated B cells. Genes which expression was detected in our mRNAseq libraries from LPS-activated B cells was used as a background gene list for GO enrichment analysis. Bubble plot sizes indicate the generality of the GO terms. Larger bubbles relate to general terms whereas smaller bubbles imply more specific GO terms
