Some identified PHAS loci of Chinese sacred lotus.

<p>The Start and End column list the start and end positions of the predicted PHAS loci in the scaffold. The TR and PR column list the total number of unique siRNAs and the number of phased unique siRNAs, respectively. The <i>P</i>-value and FDR column list the <i>P</i>-values evaluated with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113790#pone.0113790.e001" target="_blank">Equation 1</a> and the false discovery ratio using method in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113790#pone.0113790-Benjamini1" target="_blank">[35]</a>. The P.S. column lists the phase scores calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113790#pone.0113790.e007" target="_blank">Equation 2</a>. The Ref. column lists related literature of the predicted PHAS loci.</p><p>Some identified PHAS loci of Chinese sacred lotus.</p

The category of predicted PHAS loci.

<p>(a) The category of 21 nt PHAS loci. (b) The category of 24 nt PHAS loci.</p

Some LSU-rRNA loci that generate putative phasiRNAs.

<p>(a) A schematic view of phasiRNAs, and annotated genes around two predicted PHAS loci scaffold_326_1 and scaffold_326_2. (b) to (e) The read distributions and phase scores of scaffold_326_1/2, scaffold_149_1, scaffold_326_3 and sf_88_1, respectively. The legend are the same as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113790#pone-0113790-g002" target="_blank">Figure 2</a>. In the lower panel of Part b, the blue and red bars represent the phase scores of scaffold_326_1 and scaffold_326_2, respectively.</p

Putative TAS3c locus and the predicted targets of tasiRNAs in Chinese sacred lotus.

<p>(a) The sequences of the three putative TAS3 loci in Chinese sacred lotus. The red and blue regions are 5′ and 3′ miR390 complementary sites, respectively. The regions of upper case nucleotides are mature tasiRNAs that target ARF family members, or tasiARFs. The underlined region of TAS3c are a phased siRNA TAS3_D6-2(+) with position 12. (b) The 5′ miR390 binding sites on TAS3 transcripts. Only the commonly aligned nucleotides are aligned to miR390. (c) The 3′ miR390 binding sites on TAS3 transcripts. (d) The mature tasiRNAs that target ARF family members derived from TAS3a/b/c loci. (e) The reads distribution and phase score of TAS3c. Legend are the same as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113790#pone-0113790-g002" target="_blank">Figure 2</a>. The vertical dashed lines with distances of 21 nt are phase positions that start from position 10 of the 3′ miR390 site. (f) The predicted targets of tasiRNAs. The yellow and blue circle includes 7 and 8 ARF family members that are targeted by TAS3a/b and TAC3c derived tasiRNAs, respectively. (g) The complementary site of TAS3_D6-2(+) and an ARF family member, NNU_003220-RA.</p

The read distributions and phase scores of some predicted PHAS loci from NB-LRR disease resistance proteins and a MYB transcription factor.

<p>The red and green diamonds represent the number of 21 nt reads, vertical axis, that appeared at the position of the PHAS loci, horizontal axis, in the flower and leaf small RNA libraries, respectively. The vertical gray lines with distances of 21 nt are the phased positions from the position with highest phase scores of the PHAS loci. The yellow boxes in the read distribution panel represent the miRNA complementary sites. Sites pointed by miRNAs from above and under zero read line means miRNAs complement to the plus and minus strand of the predicted PHAS loci, respectively. The predicted miR2118a complementary sites are shown below the phase score panel. (a) to (c) Three potential PHAS loci, scaffold_87_1, scaffold_170_1 and scaffold_8_1, from NB-LRR disease resistance proteins. The blue sequences in the complementary sites of (a) and (b) are one of the phasiRNAs from the PHAS loci. The miR2118a site in Part c is at 176 nt 5′ side (upstream) of the PHAS locus scaffold_8_1. (d) scaffold_90_1, from an MYB90 transcription factor locus.</p

Putative TAS4 (sf_39_1, NNU_012673-RA) derives both 21 nt and 24 nt phasiRNAs.

<p>(a) The schematic view of the predicted 21 nt and 24 nt putative TAS4 and its derived phasiRNAs, as well as annotated genes. (b) The miR828 site on putative TAS4 (sf_39_1). The yellow and underlined region represent the 21 nt and 24 nt that are nearest to the miR828 site. The position pointed by an arrow is the expected phase start position that is triggered by miR828. (c) and (d) The read distribution and phase score of the 21 nt and 24 nt PHAS loci predicted. The legend are the same as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113790#pone-0113790-g002" target="_blank">Figure 2</a>. The distances between vertical gray lines in Part c and d are 21 nt and 24 nt, respectively.</p

Some predicted targets of phasiRNAs derived from the predicted PHAS loci.

<p>The M. column is the number of mismatches of the miRNA complementary sites. The names of the 21 nt and 24 nt phasiRNAs have been abbreviated to start with sf_ and sfd_, respectively.</p><p>Some predicted targets of phasiRNAs derived from the predicted PHAS loci.</p

EVO sensitize the effect of DOX without inhibiting P-glycoprotein.

<p>MCF-7 (<b>A</b>) and MCF-7/ADR (<b>B</b>) cells were pretreated with EVO and Verapamil for 12 h, and then incubated Dox (2 µM) for another 4 h, then the intracellular level of Dox was determined using flow cytometry. (<b>C</b>) Effects of EVO on the expression levels of P-gp protein in MCF-7/ADR cells. After 24 h treatment of EVO and verapamil, protein levels in cell lysates were analyzed by Western blot. GAPDH was used as an internal control. Similar results were obtained in two or three separate experiments. (<b>D</b>) After 12 h treatment, the MDR pump activities were determined using a fluorimetric MDR assay kit (Abcam). Results are expressed as mean ± SE.</p

Inhibition of Inhibitor of Apoptosis (IAPs) in MCF-7 and MCF-7/ADR cells by EVO.

<p>Whole-cell lysates were generated and immunoblotted with antibodies against XIAP, Survivin, cIAP1 and GAPDH. Similar results were obtained in two or three separate experiments.</p

Effects of different concentrations of EVO on the proliferation of Dox-sensitive MCF-7 (A) and Dox-resistant MCF-7/ADR (B) cells by MTT assay.

<p>The cytotoxicity caused by different concentrations of EVO in MCF-7 (<b>C</b>) and MCF-7/ADR (<b>D</b>) cells was determined by LDH assay. Each point represents the mean ± SE.</p