Variation in reporter gene (GFP) expression among transgenic <i>Drosophila melanogaster</i> lines.

<p>Relative expression levels of GFP were normalized to the expression of ribosomal protein 49 (<b><i>rp49</i></b>). Expression folds are calculated by comparison to line Vg1aL3, which had the lowest detectable GFP expression. Error bars represent standard errors. Letters represent statistical differences.</p

Schematic illustration of putative of regulatory elements in the promoter regions of the four <i>Culex tarsalis</i> vitellogenin genes.

<p>Schematic illustration of putative of regulatory elements in the promoter regions of the four <i>Culex tarsalis</i> vitellogenin genes.</p

Expression of GFP gene in transgenic <i>Drosophila</i> by RT-PCR.

<p>Three independent lines for each promoter were assayed. The constitutive ribosomal protein 49 gene (<i>rp49</i>) in <i>D. melanogaster</i> was examined as an endogenous control. N  =  negative control.</p

PCR primers list.

<p>PCR primers list.</p

The effect of FLASH knockdown on apoptosis was dependent on p53.

<p>(<b>A</b>) HT1080 cells were transfected with scrambled siRNA (siControl), FLASH siRNA (siFLASH) and p53 siRNA (siP53) or co-transfected with both FLASH siRNA and p53 siRNA for 48 hours. Apoptosis was then induced by incubation with 100 ng/ml FasL for an additional 4 hours. Immunoblotting using p53 antibodies showed that p53 was effectively knocked down with siP53 in the presence and absence of siFLASH. Upon stimulation with the FasL, the increase in apoptosis in cells lacking FLASH was abolished in cells depleted of both FLASH and p53. (<b>B</b>) The effect of DNA damage incurred by exposure to adriamycin on the relative intracellular level of p53 and p21. Two isogenic cell lines, HT1080 (wildtype p53) and HT1080-6TG (p53 mutant), were treated with 200 ng/ml adriamycin for the indicated times. The intracellular level of p53 and p21 was determined by immunoblotting. The level of p-Histone H2A.X (Ser139) was used to monitor the progressive DNA damage induced by adriamycin treatment. β-actin served as a loading control. (<b>C</b>) The wild type HT1080 and HT1080-6TG cells (p53 mutant) were transfected, as in panel B, with siRNA against FLASH and the scrambled siRNA (Con) for 72 hours. The intracellular levels of FLASH and MCL-1 were determined by immunoblotting. β-tubulin served as a loading control.</p

DataSheet1_Cinematic rendering improves the AO/OTA classification of distal femur fractures compared to volume rendering: a retrospective single-center study.PDF

Purpose: Correctly classifying distal femur fractures is essential for surgical treatment planning and patient prognosis. This study assesses the potential of Cinematic Rendering (CR) in classifying these fractures, emphasizing its reported ability to produce more realistic images than Volume Rendering (VR).Methods: Data from 88 consecutive patients with distal femoral fractures collected between July 2013 and July 2020 were included. Two orthopedic surgeons independently evaluated the fractures using CR and VR. The inter-rater and intra-rater agreement was evaluated by using the Cicchetti-Allison weighted Kappa method. Accuracy, precision, recall, and F1 score were also calculated. Diagnostic confidence scores (DCSs) for both imaging methods were compared using chi-square or Fisher’s exact tests.Results: CR reconstruction yielded excellent inter-observer (Kappa = 0.989) and intra-observer (Kappa = 0.992) agreement, outperforming VR (Kappa = 0.941 and 0.905, respectively). While metrics like accuracy, precision, recall, and F1 scores were higher for CR, the difference was not statistically significant (p > 0.05). However, DCAs significantly favored CR (p Conclusion: CR offers a superior visualization of distal femur fractures than VR. It enhances fracture classification accuracy and bolsters diagnostic confidence. The high inter- and intra-observer agreement underscores its reliability, suggesting its potential clinical importance.</p

siRNA silencing of FLASH expression.

<p>(<b>A</b>) HT1080 cells were transfected with FLASH siRNA and scrambled siRNA (Control) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>). After 72 hours, the extracts of the transfected cells were analyzed by immunoblotting using FLASH antibodies and as a loading control, β-tubulin antibodies. (<b>B</b>) HT1080 cells were transfected with either a scrambled siRNA (left, Control) or a specific siRNA directed against FLASH (right). The cells were fixed with cold methanol for 10 minutes after 72 hours transfection, blocked, and incubated with rabbit anti-FLASH and mouse anti-PML at 4°C overnight. After washing three times, the cells were incubated with the secondary antibodies as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#pone-0032971-g003" target="_blank">Figure 3</a>. The cell nucleus was stained with Hoechst 33342. (<b>C</b>) Flow cytometry analysis showed that after FLASH knockdown, cells were blocked in S phase. HT1080 cells were transfected with siRNA against FLASH or scrambled RNAi for 72 hours. The cells were trypsinized, washed with cold PBS, fixed with 70% ethanol, treated with RNase A and stained with 50 µg/ml propidium iodide. The DNA content was analyzed using a Becton-Dickinson FACScan cytofluorometer.</p

FLASH was found in the nucleus co-localized with NPAT.

<p>(<b>A</b>) HT1080 cells (5×10⁶) were fractionated into cytoplasmic (Cyt) and nuclear (Nuc) fractions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>). The fractions were analyzed by immunoblotting using antibodies directed against FLASH, NPAT, PARP, HDAC1 and β-tubulin. (<b>B</b>) immunofluorescence co-localization (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>) of FLASH and PML or NPAT. HT1080 cells were fixed with cold methanol for 10 minutes, blocked, and incubated with rabbit anti-FLASH and mouse anti-PML antibodies or mouse anti-NPAT antibodies at 4°C overnight. Cells were then washed 3 times and incubated at room temperature for 1 hour with a 1/2000 dilution of the secondary antibodies, Alexa Fluor 594–conjugated anti-rabbit IgG (red) and an Alexa Fluor 488–conjugated anti-mouse IgG antibody (green). The cells were also stained with Hoechst 33342 (blue).</p

Effect of FLASH knockdown on apoptotic progression.

<p>(<b>A</b>) HT1080 cells transfected with control siRNA or with siRNA directed against FLASH were stimulated with mouse anti-Fas antibody (1 µg/ml) following the standard protocol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>) for the indicated times. The cell lysates were subjected to western blotting using anti-FLASH, anti-caspase 8, anti-cleaved caspase 3 and as a loading control, anti-β-tubulin antibodies. (<b>B</b>) HT1080 cells were transfected with two different FLASH siRNAs (FLASH-1 and FLASH-2) and the scrambled siRNA (Control) for 48 hours and then treated with 100 ng/ml FasL for the indicated times. The cell lysates were subjected to immunoblotting using FLASH, PARP, caspase 8, coilin and Fas antibodies. The developed blot was scanned to determine the relative levels of active caspase-8 shown in the bar graph. (<b>C</b>) A time course showing the progression of apoptosis by immunoblotting of PARP and PARP cleavage products in control and FLASH knockdown cells following the procedure outlined in panel B. (<b>D</b>) Immunofluorescence assay of caspase-3 activation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032971#s2" target="_blank"><i>Materials and Methods</i></a>) in HT1080 cells transfected with FLASH or control siRNA for 48 hours with additional 6 hours treatment with 100 ng/ml FasL. (<b>E</b>) Light micrographs of HT1080 cells transfected with FLASH and control siRNA for 72 hours and then stimulated with FasL for 16 hours.</p

Effect of FLASH knockdown on the level of anti-apoptotic proteins.

<p>(<b>A</b>) HT1080 cells were transfected with 3 different FLASH siRNAs for 72 hours. Coilin and the scrambled siRNA served as controls. The intracellular level of FLASH, coilin, MCL-1, histone H3 and the long and short isoforms of cFLIP, cFLIP (L) and cFLIP (S), respectively, were determined by immunoblotting using the corresponding antibodies. β-tubulin served as a loading control. (<b>B</b>) Following the same protocol, MCF-10A cells were transfected with siRNA directed against FLASH or with control siRNA. Cell extracts were prepared 72 hours following transfection and the cell lyates were subjected to immunoblotting using antibodies directed against the indicated proteins.</p