Protein expression profiles of the influenza- and mock-infected MDCK cells.

<p>Cell lysates (120 µg) were separated on 13-cm (isoelectric point [pI] 4–7) linear gradient IPG strips using 12.5% SDS-PAGE. Differentially expressed protein spots are indicated with green squares. (A) Representative 2-DE gels of influenza- and mock-infected MDCK cells. T1/C: PR8-wt infected/mock infected, T3/C: rH1N1NA infected/mock infected, T4/C: rH9N2NA infected/Mock infected, T5/C: rH5N1NA infected/mock infected, T3/T1: rH1N1NA infected/PR8-wt infected, T4/T1: rH9N2NA infected/PR8-wt infected, T5/T1: rH5N1NA infected/PR8-wt infected. (B) Numbers of differentially expressed protein spots detected by 2-DE in virus-infected MDCK cells compared with mock-infected MDCK cells. The number of spots ≥0 indicated the proteins were upregulated, and the number <0 indicated the proteins were downregulated. (C) Numbers of differentially expressed protein spots detected by 2-DE in recombinant viruses compared with wild-type virus (wt-PR8)-infected MDCK cells.</p

UBE2NL protein in virus-infected or mock-infected MDCK cells at 6 h p.i..

<p>MDCK cells were infected with the viruses at MOI of 0.1 in the presence of 1 µg/ml TPCK-trypsin. After adsorption for 1 h at 37°C, the inocula were removed and the cultures were incubated for 6 h at 37°C in the maintenance media. Then, the cells were processed for indirect immunofluorescence assay, and the infected cells were detected with polyclonal antisera to UBE2NL protein and NP protein. (A) The fluorescence images (10×) of the infected and mock-infected cells at 6 h p.i. The FITC-fluorescence signal was expressed as UBE2NL protein and TRITC-fluorescence signal was expressed as the infected cells. (B) The fluorescence images (60×) of the cells infected by rPR8-H9N2NA or rPR8-H5N1NA viruses.</p

Transcriptional profiles of differentially expressed proteins in influenza virus-infected MDCKs.

<p>Total cellular RNA from MDCKs with or without influenza virus infection was subjected to real-time RT-PCR. Samples were normalized to mock-infected MDCKs using β-actin as the reference gene.</p

Western blots of representative proteins in influenza virus-infected MDCKs.

<p>The samples were prepared from MDCK cells that were virus-infected or mock-infected cells at 6 h p.i.. The β-actin protein was used as a control. (A) Western blot confirmation of differentially expressed proteins for PSMC2 (C08) and UBE2NL (C26). (B) ImageJ software analysis of the ratios of proteins changes according to Fig. 4A.</p

List of differentially expressed protein spots in MDCK cells infected with recombinant viruses and PR8-wt virus identified by MALDI-TOF/TOF.

a<p>The arrow“↑” represents the identified proteins were upregulated and the arrow “↓”represents the identified proteins were downregulated.</p

Classification of the identified proteins based on their functional annotations using Gene Ontology (GO) categories.

<p>The proteins were annotated into three main categories: cellular component, biological process, or molecular function. The Y-axis indicates the number and percentages of genes, the X-axis indicates the GO category.</p

List of differentially expressed protein spots in influenza virus-infected and mock-infected MDCKs identified by MALDI-TOF/TOF.

a<p>The arrow“↑” represents the identified proteins were upregulated and the arrow “↓”represents the identified proteins were downregulated.</p

Expression analysis of miRNAs in leaves of moso bamboo using qRT-PCR.

<p>Error bars representing the standard deviation were derived from the three experiments in triplicate.</p

Proportion of conserved miRNAs in different unearthed shoots or different portions of moso bamboo.

<p>Proportion of conserved miRNAs in different unearthed shoots or different portions of moso bamboo.</p

Discovery and Comparative Profiling of microRNAs in Representative Monopodial Bamboo (<i>Phyllostachys edulis</i>) and Sympodial Bamboo (<i>Dendrocalamus latiflorus</i>)

<div><p>Background</p><p>According to the growth pattern of bamboo, sympodial bamboo and monopodial bamboo are considered as two mainly kinds of bamboo. They have different phenotypes and different characteristics in developmental stage. Much attention had been paid on the study of bamboo cultivation, processing, physiology, biochemistry and molecular biology, which had made great progresses in the last decade, especially for the highlighted achievement of the bamboo genomics. However, there is no information available on concerning comparative profiling of miRNAs between sympodial bamboo and monopodial bamboo, which might play important roles in the regulation of bamboo development.</p><p>Methodology/Principal Findings</p><p>We identified the profiles of small RNAs using leaf tissues from one sympodial bamboo <i>i.e.</i> moso bamboo (<i>Phyllostachys edulis</i>) and another monopodial bamboo <i>i.e.</i> ma bamboo (<i>Dendrocalamus latiflorus</i>). The result showed that there were 19,295,759 and 11,513,888 raw sequence reads, in which 92 and 69 conserved miRNAs, as well as 95 and 62 novel miRNAs were identified in moso bamboo and ma bamboo, respectively. The ratio of high conserved miRNA families in ma bamboo is more than that in moso bamboo. In addition, a total of 49 and 106 potential targets were predicted in moso bamboo and ma bamboo, respectively, in which several targets for novel miRNAs are transcription factors that play important roles in plant development. More importantly, annotation of differentially expressed target genes was performed based on the analysis of pathway and gene ontology terms enrichment.</p><p>Conclusions/Significance</p><p>This study provides the first large-scale sight of discovery and comparative characterization of miRNAomes between two representative bamboos belonged to sympodial bamboo and monopodial bamboo, respectively. Although it will be necessary to validate the function of miRNAs through more experimental research in further, these results lay a foundation for unraveling the miRNA-mediated molecular processes in different kinds of bamboo.</p></div