N^pro and IRF3 co-distribute to cytoplasmic peroxisomes containing ubiquitin.

<p>(A). Cells expressing IRF3 GFP and N^pro cherry i) Control cells (CON) were stained for ubiquitin with monoclonal antibody FK2 visualized with anti-mouse Cy5. ii) Cell treated with sodium arsenate (+NaA) for 60 mins and ubiquitin was stained with monoclonal antibody FK2 and visualized with anti-mouse Cy5. iii) Cells treated with sodium arsenate (+NaA) for 60 mins and peroxisomes were stained with anti-rabbit PMP70 and visualized with anti- rabbit Cy5. (B). N^pro is stabilized and IRF3 degraded following sodium arsenate treatment. Control cells, cells expressing N^pro and cells expressing N^pro C112R mutant were untreated (−) or treated with sodium arsenate for 4 hours (+NaA). Lysates were probed by Western blot for N^pro, IRF3 and actin. Actin is shown for equal lane loading and bands were scanned and normalize with actin for N^pro levels in treated and untreated cells (bottom graph).</p

Inhibition of apoptosis by N^pro correlates with degradation of IRF3.

<p>(A). Western blotting of lysates from MEF cell expressing N^pro cherry with antibodies against N^pro, IRF3 and actin shows loss of IRF3 from cells expressing N^pro. Lane 1: Control MEF cells not expressing plasmid. Lane 2: MEF cells stably expressing N^pro mcherry. Lane 3 : MEF cells expressing N^pro C112R mcherry mutant. Lane 4: MEF cells expressing N^pro D136N cherry mutant. Graph shows relative intensities of IRF3 compared to actin for the image scanned with Scion image software <a href="http://scion-image.software.informer.com" target="_blank">http://scion-image.software.informer.com</a> (representative of Western blot repeated three times for each lane). (B). N^pro but not N^pro C112R inhibits caspase 3/7 activity. MEF cells stably expressing wild type N^pro or mutant N^pro C112R mcherry were treated with staurosporine, interferon, dsRNA (poly I:C), sodium arsenate (NaA) or hydrogen peroxide (H₂O₂) for 4 hrs. Fold increase in caspase activity was normalized to negative (untreated cells) for each cell line and analyzed with a two-sided t-test with unequal variance relative to the untransfected cells for each cell line, and significance values are: bars marginally significant (0.05–0.1), * significant (0.01–0.05), ** highly significant (0–0.01) (n = 5 to 8). The table shows significance of drug treatment relative to control treated cells for cells expressing N^pro WT or N^proC112R and analysis was conducted using similar tests as described above.</p

Additional file 1: Table S1. of Identification of miRNAs with potential roles in regulation of anther development and male-sterility in 7B-1 male-sterile tomato mutant

List of the identified known miRNAs in WT and 7B-1 anthers. Table S2. List of the new miRNAs identified from WT anthers. Table S3. List of the new miRNAs identified from 7B-1 anthers. Table S4. List of miRNA-target cleavage sites. Table S5. List of tasiRNAs and their predicated target genes. Table S6. List of the identified TAS3-derived tasiRNAs from WT library. Table S7. List of the identified TAS3-derived tasiRNAs from 7B-1 library. Table S8. List of the primers used for RT-qPCR analysis. Table S9. List of the primers used for 5′-RACE analysis. Table S10. List of the DIG-labeled oligo-probes used for in situ hybridization. (DOCX 676 kb

N^pro co-distributes with peroxisomes marker PMP70.

<p>(A). Peroxisomes were localized by immunostaining with anti-peroxisome marker PMP70 in MEF cells expressing N^pro cherry i) control cells (CON) ii) treatment with sodium arsenate (+NaA) for 4 hours iii) Digital rendering using Imaris software is shown for the images in ii) (scale bars are 5 um). (B). Statistical analysis of average number of N^pro puncta per cell and average number co-distributing with PMP70 after sodium arsenate treatment for 4 hours. (C). Boxplot: distribution of the fraction of N^pro distributing with PMP70 compare to total number of N^pro granules. A Welch two sample t-test was applied, two-sided, not paired, (assuming that the variances are unequal) and showing t = 0.5867, df = 25.712, p-value = 0.5625 with the lower end of the box = 25% of the data, bold line in the middle = median, Upper end of the box = 75%, lower whisker = 5%.</p

N^pro is recruited to mitochondria after cell stress and protects from apoptosis.

<p>(A). MEF cells were transfected with Bax-GFP alone (a–f) or tBid-GFP alone (g–j) or N^pro cherry alone (k–n) and either untreated (No NaA, left panel) or treated with sodium arsenate for 4 hours (+NaA, right panel). Cells were incubated with Mitotracker (MT) CM Ros (b,e,i) or Green FM (m) for 30 mins before fixation. (B). Protection from apoptosis induced by sodium arsenate requires IRF3. MEF cells co-expresssing Bax-GFP and either wild type N^pro-cherry (a–b) or mutant N^pro C112R-cherry (c–d) were treated with sodium arsenate (+NaA). MDBK primary cells, transfected with Bax GFP (e–f) alone or infected with BVDV overnight and then transfected with Bax GFP (g–h), were treated with sodium arsenate (+NaA) for 4 hours. BVDV was detected with bovine hyperimmune serum V182 and detected with Cy3 rabbit anti bovine antibody (red) and DNA was stained with DAPI.</p

N^pro rapidly redistributes with IRF3 to mitochondria and cytoplasmic puncta following induction of stress.

<p>(A). MEF cells co-expressing N^pro -cherry (red) and IRF3-GFP (green) were treated with sodium arsenate (+NaA) for 30 minutes and examined by confocal microscopy. (B). Treatment of MEF cells co-expressing N^pro-cherry and IRF3-GFP for 60 minutes.</p

Analysis of SINV matching reads indicate degradation.

<p>(A) Nucleotide variation across the genome (x axis) at 0, 4 and 6 hpi on both positive and negative strands is indicated by the number of unique sRNAs (0 to 300 and 0 to −300, respectively) which vary at a given position (y axis). This analysis indicates a high similarity of the Sindbis variants present in the cell to the reference sequence. The open reading frames are indicated by black boxes at the top. (B) Variation of expression level (log2 scale) for SINV matching reads. The figure shows the distribution of viral reads along the SINV genome (x axis). Positive values on y axis indicate the abundance of reads mapping to the positive strand of the virus, negative values indicate the abundance of reads mapping to the negative strand. Black represents the 4hpi reads, grey represents the 6hpi reads. (C) Variation of p Value presented on the y axis for a <i>x</i>² significance test on the size class distribution compared to a random uniform distribution for windows of length 100 nt along the SINV genome (x axis). Black represents 4hpi and grey represents 6hpi sRNA samples.</p

Size class and complexity distributions of sequencing reads.

<p>Size distribution and complexity are shown for (A) all reads, (B) reads mapping to the Human genome, (C) reads mapping to the SINV genome. Subplots A1, B1 and C1 show the read number for 21–24 mers at 0, 4 and 6hpi. Subplots A2, B2 and C2 show the complexity for each size class from 17–27 mers, where the complexity is the ratio of non-redundant reads to redundant reads.</p

Infection and replication of SINV in HEK293 cells.

<p>(A). Northern blot showing SINV full length genome (49 S) and subgenomic (26 S) positive strand RNA rapidly accumulates in HEK293 cells from 2hpi and increasing through 8 hpi. The probe consisted of P32 end-labelled primers at positions 7568–7631 in the genome. The 28 S and 18 S RNA bands stained with ethidium bromide are displayed to demonstrate equal loading. (B) Immunostaining of SINV in HEK293 cells using rabbit anti E2 glycoprotein antibody detected SINV E2 translation increasing over 4, 6 and 8 hpi with over 95% of cells infected with well-defined replication centres (visualised with an anti-rabbit secondary antibody labelled with Alexa488 and nuclei are shown with DAPI).</p

Additional file 7: of Profile and functional analysis of small RNAs derived from Aspergillus fumigatus infected with double-stranded RNA mycoviruses

Characteristics of Folded-1 (PDF 787 kb