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Tissues 36-44 h APF were blocked in PBS + 0.3% Triton X-100 + 1% BSA (PAT) overnight. Tissues 24-36 h APF were blocked in PBS + 0.1% Triton X-100 + 1% BSA for 1 h. Pupal cuticle was removed from wings after fixation.

Pupae, staged from white prepupae (0 h) at 25Р Р†Р’ВР Р‰, were dissected and fixed with 4% paraformaldehyde in 1Р вЂњРІР‚вЂќ PBS for 30 min at room temperature.

6 H), and very low levels of CycB in wings and eyes Fig. In contrast, cells expressing E2F + Rca1 together accumulated CycA in wings (Fig. S3), despite expressing high levels of cycA, gem, stg, and cycB transcripts by microarray (see supplemental dataset and JCB DataViewer). This is consistent with our finding that E2F1-DPoverexpressing cells do not accumulate the known APC/C targets CycA, Geminin (Gem Zielke et al., 2008), CycB, or Stg (based on a Stg-GFP fusion protein) after prolonged exit in wings or eyes (40-44 h APF Fig. We hypothesized that high APC/C activity after exit could limit E2F-induced bypass of cell cycle exit by degrading key E2F targets. (B) A group of 162 genes was up-regulated >1.75-fold (>log 2 0.82) by E2F activity at one or both time points APF but was not significantly increased in Dp Arrays for E2F and Dp Р С•Р вЂљР вЂ /Р С•Р вЂљР вЂ at 24 and 36 h were highly similar with a correlation coefficient of 0.74. Typical group transcripts are up-regulated in E2F-expressing and Dp Р С•Р вЂљР вЂ /Р С•Р вЂљР вЂ wings at 24 and 36 h APF, whereas other transcripts are repressed or inversely regulated. Representative genes from each major cluster are listed on the right.

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Transcripts were clustered using Genesis software for hierarchical clustering. Gray bars indicate transcripts that were removed from analysis because of high variability among independently replicated experiments (n = 4 for E2F n = 3 for Dp Р С•Р вЂљР вЂ /Р С•Р вЂљР вЂ ). A level of p log 2 Р вЂ™Р’В± 0.6) at one or more time points are shown. The Spearman rank correlation coefficient was used to analyse the correlation between the radiographer's experience in performing X-rays and collimation quality. Student's t test was used to calculate differences between DAPs. Optimal 0.32 * 0.39 * Sub-optimal 0.68 0.61 1-2 parts exposed Slightly reduced 0.48 0.49 3-4 parts exposed Moderately reduced 0.17 0.08 5-6 parts exposed Markedly reduced 0.02 0.04 >7 parts exposed Severely reduced 0.01 0.00 rected for continuity) was used to calculate differences between the hospitals, as well as between optimal and suboptimal images with respect to age, gestation, number of tubes and catheters, radiographer, and the number of years' experience. The readers resolved any differences by consensus. Radiographic errors were recorded on individual tick sheets and the information was captured in an Excel spreadsheet ( Microsoft, Redmond, WA). The image quality in terms of correct collimation was graded arbitrarily as follows: 0 points = optimal image quality 1-2 points = slightly reduced 3-4 = moderately reduced 5-6 = markedly reduced and >7 points = severely reduced Fig. Inappropriate exposure of the arms was assumed when more than the diametaphyseal junction of the proximal humerus came within the field of view: part of the diaphyseal humerus (1 point) entire humerus (2 points) part of the forearm (1 point) entire forearm (2 points) hand (1 point). Exposure of the cranial structures was considered inappropriate when the collimated field included more than the tip of the mandible (1 point). Inappropriate exposure of abdominal viscera was assumed when the caudal imaging field extended below the level of L1/2 (1 point).

A grading system was used to measure image quality in terms of correct collimation. The readers performed their evaluations according to the European Guidelines on Quality Criteria for Diagnostic Radiographic Images in Paediatrics.

They determined the most superior and inferior parts of the body, as well as the lateral structures, which had been included within the boundaries of collimation on each chest X-ray. Two experienced radiologists (JCS and IK-S) evaluated the image quality, based on the exposure of non-thoracic structures.

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