Comparison of the DCBB set of genes with the Ciliary proteome and Ciliome databases

Venn diagram presenting the overlaps between the three datasets: the cilia proteome [46,48]; the ciliome [47,49], and the DCBB (Additional data file 2). Asterisks indicate this study. Note that only 412 common genes are found in the three datasets. The number of genes also found in the 1,462, 412 or 83 X-box gene lists (Table 4), respectively, are noted in parentheses. The numbers of genes selected in the different studies to construct each dataset are given in Additional data file 3.<p><b>Copyright information:</b></p><p>Taken from "Identification of novel regulatory factor X (RFX) target genes by comparative genomics in species"</p><p>http://genomebiology.com/2007/8/9/R195</p><p>Genome Biology 2007;8(9):R195-R195.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2375033.</p><p></p

Additional file 2: of Characterization of the human RFX transcription factor family by regulatory and target gene analysis

Hierarchical clustering, expression plots and top 10 tissues, primary cells and cell lines of RFX TSS locations. Hierarchical clustering of 30 RFX TSS locations (with shorthand p for promoter) based on expression values (TPM) across 135 human tissue samples, using a 1-Pearson correlation distance measure and average linkage method, as computed by the pvclust R package with nboot = 1000 with the numbers representing approximately unbiased (au) p-values (Suzuki and Shimodaira, 2006). Tissue clusters are color-coded and represent the groups of tissues with the highest overall expression values: immune system (teal), gastrointestinal tract (purple), testis (green), brain and spinal cord (red), and two minor clusters, uterus and lung (black). RFX TSS locations without color code have low expression values (TPM < 5). This is followed by the expression profiles of 30 RFX TSS locations in human tissues, primary cells and cell lines, whereby for every one of the eight human RFX genes (1–8), summarized TSS profile data are presented vertically (“top-down”), starting with the a tissue plot, followed by a table of the top 10 tissues, a table of the top 10 primary cells and a table of the top 10 cell lines (highest expression levels are listed first, respectively). The tissue plot is the expression level in log (base 10) TPM against tissues that are sorted from the highest to the lowest expressed from 135 tissues, whereby the plot only includes the first 100 tissues. The arbitrary unit for detection of expression is tags per million (TPM) as defined by FANTOM5. We consider TPM < 5 to be lowly expressed and TPM < 1 to be background noise. (PDF 3276 kb

Additional file 4: of Characterization of the human RFX transcription factor family by regulatory and target gene analysis

Detailed candidate RFX regulator oPOSSUM3 scanning results using JASPAR 2016 core vertebrate TF binding profiles. Transcription factor binding sites (TFBS) scanning results from oPOSSUM3 within the promoter and enhancer regions of RFX1–8 using the CORE vertebrate TF binding profiles in JASPAR 2016. Included are the DNA regions that were considered as foreground and the following TF binding site details: SP2 (specificity protein 2) (JASPAR profile MA0516.1) and ESR1 (estrogen receptor alpha) (MA0112.3). (XLSX 50 kb

Additional file 1: of Characterization of the human RFX transcription factor family by regulatory and target gene analysis

Detailed expression values (TPM) for RFX TSS locations. Expression values in tags per million (TPM) for all 30 RFX TSS locations in all 889 biological samples and their categorization into tissues (135), primary cells (473 donor replicates and 170 merged replicates from the average TPM value of the donor replicates), cell lines (255) and time courses (26). (XLSX 355 kb

Additional file 5: of Characterization of the human RFX transcription factor family by regulatory and target gene analysis

Ct levels of qRT-PCR, used for validation of candidate RFX regulators by siRNA knockdown. Individual Ct levels with automatic threshold obtained on an AB7500 Fast machine for SP2 and ESR1 as candidate RFX regulators and their respective test siRNA and scrambled (Scr) control siRNA knockdown data on RFX genes (RFX1, RFX2, RFX3, RFX5, RFX7) and the two reference genes (HPRT1, HSPCB). (XLSX 33 kb

Candidate ABCE1-interacting proteins.

<p>HEK293 cells expressing V5-tagged ABCE1 and mock-transfected cells were subjected to IP with anti-V5 antibody. The protein content of the obtained immune complexes was analyzed using LC-MS/MS. Potential ABCE1 binding partners (proteins present in at least two experimental samples with experimental and control sample intensity ratio > 2, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116702#pone.0116702.s004" target="_blank">S1 Table</a>) that could be linked to RNA silencing and have putative homologs in <i>A</i>. <i>thaliana</i> and <i>C</i>. <i>elegans</i> (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116702#pone.0116702.s005" target="_blank">S2 Table</a>) were grouped according to their biological functions: epigenetic regulation (blue), transcription/transcription regulation (orange), RNA processing (green), mRNA surveillance (pink) and RNA silencing (brown). Four proteins were categorized into two groups and are therefore marked differently: EXOSC4—epigenetic regulation and transcription/transcription regulation, PSIP1—RNA processing and transcription/transcription regulation, SMARCA5—transcription/transcription regulation and epigenetic regulation, SUPT16H—epigenetic regulation and transcription/transcription regulation. ABCE1 is depicted in yellow.</p

Northern blot analysis showing the suppression of <i>GFP</i> RNA silencing in <i>N</i>. <i>benthamiana</i> by ABCE1.

<p><b>(A)</b> GFP-transgenic <i>N</i>. <i>benthamiana</i> 16c leaves were infiltrated with <i>A</i>. <i>tumefaciens</i> harboring pBin61-GFP together with <i>A</i>. <i>tumefaciens</i> carrying pBin61-AtRLI2 or pBin61-ABCE1 or empty vector (pBin61), as indicated on the upper part of the panel. Total RNA was extracted from the infiltrated patches and analyzed by northern blot. Levels of GFP mRNA in the patches infiltrated with pBin61-GFP/pBin61-AtRLI2 or pBin61-GFP/pBin61-ABCE1 were higher than in the case of pBin61. 16c indicates non-infiltrated leaf. GFP mRNAs were detected using [α-³²P] UTP-labeled antisense GFP transcripts. Ethidium bromide staining of rRNA was used as loading control. <b>(B)</b> Total RNA was extracted from the infiltrated patches and analyzed by northern blot as indicated before. GFP siRNA levels were reduced in the presence of AtRLI2 or ABCE1 compared to the control (pBin61). 16c indicates non-infiltrated leaf. For the detection of GFP siRNAs [γ-³²P] ATP end-labeled GF-probe was used. U6 stands for U6 snRNA used as loading control. <b>(C)</b> pBin61-GFFG was infiltrated as RNA silencing inducer instead of pBin61-GFP. The northern blot analysis of GFP mRNAs was performed as in (A) and shows higher levels in the presence of AtRLI2 or ABCE1 than in the case of the empty vector. <b>(D)</b> The accumulation of GFP siRNAs in the patches infiltrated with pBin61-GFFG/pBin61-AtRLI2, pBin61-GFFG/pBin61-ABCE1 or pBin61-GFFG/pBin61 was analyzed as in (B). GFP siRNA levels were reduced in the presence of AtRLI2 or ABCE1 as compared to the control (pBin61).</p

ABCE1 suppresses RNAi of <i>GFP</i> in <i>C</i>. <i>elegans</i>.

<p><b>(A)</b> Schematic representation of the <i>in vivo</i> RNAi inhibition assay—the transgenes expressed and the GFP status are indicated. (<b>B)</b> Representative photomicrographs of the posterior part of animals expressing the NLS::GFP reporter in body wall muscles, treated with <i>GFP</i>-specific RNAi for 24 h at 20°C; control RNAi—empty vector. (<b>C)</b> Representative photomicrographs of the posterior part of animals expressing <i>C</i>. <i>elegans</i> ERI-1 and the NLS::GFP reporter in body wall muscles, treated with <i>GFP</i>-specific RNAi for 24 h at 20°C; ERI-1(+)—animals carrying the <i>myo-3</i>::<i>eri-1</i> transgene; ERI-1(-)—animals not carrying the <i>myo-3</i>::<i>eri-1</i> transgene. (<b>D)</b> Representative photomicrographs of the posterior part of animals expressing ABCE1 and the NLS::GFP reporter in body wall muscles, treated with <i>GFP</i>-specific RNAi for 24 h at 20°C. ABCE1(+)—animals carrying the <i>myo-3</i>::<i>ABCE1</i> transgene; ABCE1(-)—animals not carrying the <i>myo-3</i>::<i>ABCE1</i> transgene. (<b>E)</b> Relative GFP fluorescence intensity of the NLS::GFP reporter in worms expressing ERI-1 and ABCE1 and treated with <i>GFP</i>-specific RNAi for 24 h at 20°C. The ratio of the GFP signal intensity in worms expressing ERI-1 or ABCE1 and the NLS::GFP reporter compared to reporter alone is presented. Results from a representative experiment are shown (n > 100). Error bars represent the 95% confidence interval for the mean. Asterisks denote a statistically significant increase of the GFP signal intensity in worms expressing ERI-1 or ABCE1 and the NLS::GFP reporter as compared to the reporter alone (p < 0.0001 by two-tailed Student’s <i>t</i>-test), showing that ERI-1 and ABCE1 are able to suppress <i>GFP</i> RNAi.</p

ABCE1 suppresses RNAi mediated silencing of <i>ULK3</i> in HEK293 cells.

<p>FLAG-tagged ULK3 was expressed in HEK293 cells in combination with empty vectors pcDNA3.1 and pSUPER or with siRNA(ULK3) or scrambled siRNA(X) and plasmids encoding either ABCE1 or P19 proteins. Cells were analyzed 30 h post-transfection. <b>(A)</b> FLAG-tagged ULK3 and actin (loading control) were detected by western blotting. The blot shown is representative for three independent experiments. ABCE1 and P19 were able to increase ULK3 expression levels in silenced cells. ABCE1 and P19 did not have any significant effect on ULK3 expression level in siRNA(X) transfected (non-silenced) cells. Molecular masses (in kDa) are shown on the right. <b>(B)</b> Quantification of relative ULK3 expression levels derived from three independent experiments. ULK3 expression levels were normalized to actin expression levels. The means relative to the levels of non-silenced ULK3 (cells transfected with empty vectors) are shown. Error bars indicate standard deviations. ABCE1 and P19 rescued ULK3 expression level significantly (*p = 0.0835, **p = 0.0461).</p

Additional file 1: of Fully automated, inline quantification of myocardial blood flow with cardiovascular magnetic resonance: repeatability of measurements in healthy subjects

Figure S1. Correlation by slice (A) rest (B) stress. Trend line represents line of perfect fit. (DOCX 119 kb