ARNTL regulates p21^CIP1 expression.

<p>a) Knockdown of ARNTL inhibits radiation induced p21^CIP1 induction. U2OS cells were infected with the shRNA vectors as indicated. Cells were seeded and irradiated with 20 Gy of γ-radiation. After o/n incubation cells were lysed and lysates were subjected to western blot using antibodies for p53, CDK4 (control) and p21^CIP1. b) Knockdown of ARNTL can also rescue a p19-induced cell cycle arrest. U2OS cells were infected with the indicated shRNA vectors followed by a super-infection with p19ARF-RFP virus. Cells were seeded and incubated for three weeks. After three weeks the infected cells were fixed and stained. c) Knockdown of ARNTL in U2OS cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g005" target="_blank">Fig 5b</a>) was quantified by QRT-PCR. d) ARNTL knockdown is not involved in p53 independent p21^CIP1 induction. HCT116 wt and p53^−/− cells were infected with knockdown vectors targeting p53, p21^CIP1 and ARNTL. Cells were treated with 0.5 µM PXD101 for 16 hrs. Cells were then lysed and lysates were subjected to western analysis for p53, CDK4 (control) and p21^CIP1. e) Quantification of p21 protein levels in the western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g005" target="_blank">figure 5c</a> using IMAGE J software.</p

shRNA barcode screen identifies mediators of the p53 dependent cell cycle arrest.

<p>a) Schematic outline of the BJtsLT genetic screen. BJtsLT cells were infected with the NKI shRNA library and were either left at 32°C or shifted to 39°C. After 7 days the cells at 32°C had reached confluency and were harvested. Cells at 39°C were harvested after 23 days after which they had formed visible colonies. b) Analysis of the relative abundance of shRNAs recovered from the BJtsLT barcode experiment. Data are normalized and plotted as <i>M</i>, the ²log (ratio Cy5/Cy3), versus <i>A</i> (²log(√intensity Cy3×Cy5)). The data are the average of two independent hybridization experiments performed in duplicate with reversed colour. A red box is drawn around the top 100 enriched shRNAs at 39°C. c) Schematic overview of selection criteria used to select hits from the shRNA barcode screen for further validation.</p

Colony formation ARNTL, RBCK1 and TNIP1 shRNA vectors.

<p>Cells were infected with shRNA vectors targeting ARNTL, RBCK1 and TNIP1 and control shRNA vectors targeting GFP, p53, 53BP1 and p21. Cells were infected at 32°C and shifted to 39°C 2 days after infection. After three weeks culture at 39°C, the cells were fixed and stained.</p

Knockdown of ARNTL, TNIP1 and RBCK1 prevents p21^CIP1induction in BJtsLT cells.

<p>BJtsLT cells were infected at 32°C and shifted to 39°C for colony formation. After 14 days of culturing at 39°C cells were harvested, protein lysates were prepared and subjected to western blot for p53, CDK4 (control) and p21^CIP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g004" target="_blank">Figure 4a–c</a>). Additionally, total RNA was isolated and used for QRT-PCR for p21^CIP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004798#pone-0004798-g004" target="_blank">Figure 4d</a>).</p

Barcode identified shRNA vectors suppress protein and mRNA levels of their targets.

<p>a) QRT-PCR for ARNTL in BJtsLT cells. BJtsLT cells were infected with indicated shRNA vectors. Samples for RNA isolation were taken 8 days after shift to 39°C. b) QRT-PCR for RBCK1 in BJtsLT cells. BJtsLT cells were infected with indicated shRNA vectors. Samples for RNA isolation were taken 8 days after shift to 39°C. c) QRT-PCR for TNIP1 in BJtsLT cells. BJtsLT cells were infected with indicated shRNA vectors. Samples for RNA isolation were taken 8 days after shift to 39°C. d) Flag-ARNTL together with the shRNA vectors targeting ARNTL were transiently transfected in Phoenix cells. Extracts were immunoblotted using Flag and CDK4 (control) antibodies. e) BJ cells were infected with the indicated shRNA vectors and Extracts were immunoblotted using TNIP1 and CDK4 (control) antibodies.</p

List of shRNA vectors that were selected by the criteria as in figure 1c. Depicted are shRNAs that were used in validation experiments.

<p>Under rescue; + means a validated shRNA, − means not validated. <i>M</i> indicates the ²log (ratio Cy5/Cy3), A the ²log(√intensity Cy3×Cy5).</p

Primary enucleation and OAC treatment in unilateral advanced eyes.

<p>The number of eyes that were primarily enucleated (black) or treated with OAC (gray) per year.</p

Ophthalmic artery chemosurgery for eyes with advanced retinoblastoma

<p><i>Background</i>: Surgical removal of one or both eyes has been the most common way to treat children with retinoblastoma worldwide for more than 100 years. Ophthalmic artery chemosurgery (OAC) was introduced 10 years ago and it has been used as an alternative to enucleation for eyes with advanced retinoblastoma. The purpose of this report is to analyze our 9-year experience treating advanced retinoblastoma eyes with OAC.</p> <p><i>Materials and methods</i>: Single-arm retrospective study from a single center of 226 eyes with eyes of retinoblastoma patients with advanced intraocular disease defined as <i>both</i> Reese-Ellsworth (RE) “Va” or ”Vb” <i>and</i> International Classification Retinoblastoma (ICRb) group “D” or ”E” (COG Classification). Ocular survival, patient survival, second cancers, and electroretinography (ERG) were assessed.</p> <p><i>Results</i>: Ocular survival at five years for these advanced eyes was 70.2% (95% confidence interval, 57.3%–79.8%). When eyes were divided into groups either by RE classification or ICRb, no significant differences in ocular survival were seen. Ocular survival was significantly better in naïve compared to non-naïve eyes (80.2% vs 58.4%, <i>p</i> = 0.041). The ERG distribution was very similar before and after OAC treatment for the patient population that did not receive intravitreal chemotherapy. Three patients (1.5%) have developed metastatic retinoblastoma (previously reported) and were successfully treated (no deaths).</p> <p><i>Conclusion</i>: Using OAC for advanced eyes (the majority of such eyes have been enucleated in the past) enables 70% 5-year ocular survival. Treated eyes have a similar ERG distribution before and after treatment. No patient has died of metastatic retinoblastoma.</p

Drug Combinations Used Per OAC Treatment.

<p>Drug Combinations Used Per OAC Treatment.</p

Kaplan Meier Ocular Survival Curve.

<p>One hundred and twenty eyes of 60 patients who underwent bilateral ophthalmic artery chemosurgery (Tandem Therapy) were assessed for enucleation. Ocular survival was 99.2% at one year, 96.9% at 2 and 3 years, and 94.9% for years 4 through 7.</p