Deficient S phase checkpoint regulation upon etoposide treatment in the absence of CCDC6.

<p>(<b>A</b>) HCT116 cells were treated with 20 µM etoposide and cells were harvested at predetermined time points for cell cycle analysis. In the absence of CCDC6, no S phase accumulation is observed and the transition to G₂ phase is accelerated. One representative experiment is shown, out of three performed. (<b>B</b>) Concomitant apoptotic cell death was quantified by measuring the subG₀/G₁ DNA content. CCDC6 knock down cells showed higher levels of apoptosis, at earlier time point, in comparison to the control, in response to genotoxic stress upon etoposide treatment. (<b>C</b>) The percentage of cell survival was assessed by gating for PoPRO and 7-AAD negative cells. CCDC6 knock down resulted in lower cell survival upon etoposide induced genotoxic stress. The assays were performed in triplicates.</p

CCDC6 knock down results in altered cellular localization of CDC25C and accelerated G₂/S transition upon etoposide-mediated genotoxic stress.

<p>Control and CCDC6 knock down HCT116 cells were treated with etoposide (20 µM). (<b>A</b>) Cell lysates of HCT116 cells treated with etoposide (20 µM) for 2, 4, 8, 12 and 24 hours and mock control treated with DMSO vehicle were resolved on a SDS-PAGE and probed for 14-3-3σ and CDC25C. 14-3-3σ protein levels were down-regulated in the absence of CCDC6 protein expression and the CDC25C protein level regulation was altered. (<b>B</b>) Cells grown on cover slips were exposed to etoposide for 4, 8, 12, 24 hours, fixed and stained for CDC25C. In mock cells, CDC25C is kept in the cytosol upon etoposide treatment at 8 and 12 hours but is localized in the nucleus in the absence of CCDC6. (<b>C</b>) Cells exposed to etoposide for 12 hours were co-stained for CDC25C and 14-3-3σ. CDC25C is kept in the cytosol upon etoposide treatment and exhibits co-localization with 14-3-3σ (seen in yellow) but enters the nucleus in the absence of CCDC6.</p

CCDC6 knock down alters proliferation rate and increases cell death <i>in vitro</i>.

<p>Cells were transduced using lentivirus, expressing two different small hairpins for CCDC6, labeled as sh1 and sh2 and cultured for 48 hours. The same viral vector was applied as control (mock: mock transduced). (<b>A</b>) Western blot analysis using anti-CCDC6 mouse monoclonal antibody demonstrated the efficient knock down of CCDC6 protein expression. Growth curves were performed in triplicates using trypan blue dye exclusion for counting the alive (<b>B</b>) and the dead cells (<b>C</b>). Decreased proliferation rate and increased cell death was observed in the absence of CCDC6. (<b>D</b>) The subG₀/G₁ population, as measured by flow cytometry, is indicative of apoptosis and is significantly increased following CCDC6 knock down. The percentage of survival was calculated for each time point by excluding both early apoptotic and dead cells. (<b>E</b>) Apoptotic cell death was analyzed by Po-PRO and 7-ADD staining. The Po-PRO single-positive cells are early apoptotic while the double positive stained cells for Po-PRO and 7-AAD are late apoptotic and dead cells. All assays were performed in three independent experiments.</p

Normal Cell cycle progression is altered upon CCDC6 knock down.

<p>Cell cycle analysis was performed using propidium iodide (PI) staining and measuring the DNA content, at the indicated time points, starting 48 hours after transduction. Cell cycle was analyzed with FlowJo software and Jean-Fox algorithm. In all time points both in HCT116 (<b>A</b>), (<b>B</b>) and HeLa (<b>C</b>), (<b>D</b>) the percentage of cells in the S phase is reduced upon knock down of CCDC6 in comparison to the control (mock). One, out of three, representative experiment is shown. (<b>E</b>) HCT116 cells were synchronized by serum starvation for 48 hours followed by restimulation with 5% of FCS. CCDC6 knock down resulted in incomplete arrest at G₁ and not total synchronization, as the control cells. 14 hours after serum stimulation the majority of the control cells are in S phase while CCDC6 knock down cells demonstrated a delay in S phase entering. 4 hours later, control and CCDC6 knock down cells showed the same profile, suggesting shorter duration of S upon CCDC6 knock down. 24 hours later, control cells were cycling normally and CCDC6 knock down cells exhibited a delay in completing G₂ phase and re-entering G₁.</p

Classification of genes that are induced or repressed following pathogenic BmCPV infection: comparison among different relevant studies.

<p>Only genes confirmed by qRT-PCR (more than 2-fold difference in response) are shown. Induced and repressed genes are marked with up and down arrows respectively. Empty cells indicate that the particular gene was not tested by qRT-PCR for differential expression in the particular study. Abbreviations: SH: subtractive hybridization, MA: microarray, DS: deep sequencing. P50, 4008 and Daizo refer to silkworm strains.</p><p>Classification of genes that are induced or repressed following pathogenic BmCPV infection: comparison among different relevant studies.</p

Detection of BmCPV <i>polyhedrin</i> by RT-PCR in persistently and pathogenically infected silkworm tissues and eggs.

<p><i>Polyhedrin</i> RT-PCR was performed using samples derived from larvae persistently and pathogenically infected (a) at the 2^nd or (b) the 4^th instar (2c, 2inf, 4c and 4inf samples), as well as from Daizo and P50 silkworm strain-derived eggs. <i>Polyhedrin</i> PCR product with a size of 689 nucleotides was obtained after 40 cycles of PCR. Abbreviations: BW: body wall, MG: midgut, MGC: midgut content, F: feces, (+): positive control (BmCPV polyhedra), (-): negative control (water sample).</p

Expression levels of RNAi-related genes from pathogenically infected midguts as analyzed by deep sequencing and validated by qRT-PCR.

<p>Indicated is the fold change in expression of selected genes between persistent and pathogenic infection as obtained by qRT-PCR on 2^nd instar samples (qPCR-2) or by deep sequencing on both 2^nd and 4^th instar samples (DS-2 and DS-4, respectively). Please note that fold changes are expressed as log2 values (a 2-fold up- or down-regulation corresponds to a log2 value of 1 or -1, respectively). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121447#pone.0121447.t005" target="_blank">Table 5</a> and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121447#pone.0121447.ref064" target="_blank">64</a>] for further explanation on gene identity and function.</p

Gene ontology (GO) analysis of differentially expressed genes in pathogenically infected larvae.

<p>Genes that were differentially expressed during pathogenic infection at both 2^nd and 4^th instar developmental stages were analyzed using GO tools and categorized according to biological process, molecular function and cellular component classes. The numbers of genes that could be assigned to the different categories are indicated.</p

Differentially expressed genes following pathogenic infection in midgut, as determined by deep sequencing analysis.

<p>Numbers of up-regulated and down-regulated genes are indicated, after comparison of RPKMs of identified genes between 2inf and 2c libraries (2^nd instar), and between 4inf and 4c libraries (4^th instar). The numbers of genes that were up-regulated or down-regulated in both developmental stages are indicated in the section of the two diagrams.</p

Relative RPKMs of genes coding for core RNAi factors.

<p>Ratios of RPKM values from pathogenically versus persistently infected midgut tissue of 2^nd and 4^th instar larvae obtained by deep sequencing analysis (2c, 2inf, 4c and 4inf samples) are presented for selected genes belonging to the miRNA, siRNA and piRNA pathways. Genes presenting higher than 1.5-fold up- or down-regulation are marked with bold letters.</p><p>Relative RPKMs of genes coding for core RNAi factors.</p