horizondiscovery.com p53 APPLICATION NOTE Žaklina Strezoska, Horizon Discovery, Lafayette, CO, USA Tamara Seredenina, Siena Biotech, Siena, Italy

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1 APPLICATION NOTE Silencing of expression with Accell sirna causes inhibition of the DNA damage response in IMR-32 neuroblastoma cells and protects primary cortical neurons from β-amyloid toxicity Žaklina Strezoska, Horizon Discovery, Lafayette, CO, USA Tamara Seredenina, Siena Biotech, Siena, Italy Introduction Neuroblastoma cell lines and primary neuronal cultures are commonly used as cellular model systems for studying cancer and neuronal development as well as being relevant cells for the study of neurodegenerative diseases. However, most neuroblastoma cell lines and primary neuronal cells suffer from low transfection efficiency due to the refractory nature of the cells to lipid-based transfection reagents. As such, application of small interfering RNA (sirna) for inducing RNA interference (RNAi) has limited utility in these cell types, thus hindering the development of functional assays for screening and discovery of novel disease-relevant genes. Dharmacon Accell sirna reagents enable efficient delivery in a wide range of cell lines and primary cells. Accell sirna reagents carry a novel chemical modification pattern that facilitates the delivery of sirna without a need for transfection reagents. To demonstrate the utility of Accell sirna reagents in neuronal cells, the effects of silencing the expression of was examined. is a tumor suppressor protein that regulates the cellular response to different DNA damaging or cellular stress agents by controlling the expression of a wide variety of genes involved in cell growth, repair and survival or causing the cell to enter apoptosis if damage can not be repaired (Figure 1). Here we describe how application of Accell sirna enabled the development of a high content assay for multiplex analysis of and p21 expression in IMR-32 neuroblastoma cells, as well as a whole-culture cell viability assay in IMR-32 and primary rat cortical neurons. The ability to modulate gene expression in neuronal cell lines and primary neurons using Accell sirna demonstrates potential new opportunities for functional genomic sirna screens in the field of neuroscience. Stress p21 DNA damaging agents (such as camptothecin) neurotoxins (such as amyloid-β) UV, hypoxia, etc. GAD45α σ Bax Apaf-1 Casp- 9 Cell cycle arrest/genetic repair Cell survival P Mdm2 Apoptosis Death of damaged cells Figure 1. is a a tumor suppressor protein that mediates cellular response to different stress agents. Cell exposure to damaging agents induces rapid increase of protein levels in the nucleus, leading to the induction of its transcription targets controlling cellular responses such as cell cycle arrest, repair and cell survival (p21 Waf1 Cip1, GAD45α, σ) and/ or apoptosis (for example Bax, Apaf1, Casp-9).

2 Results Efficient target mrna knockdown in IMR-32 by Accell sirna In order to examine whether the Accell sirna delivery protocol can be applied to IMR-32 neuroblastoma cells, several Accell sirna controls were tested for silencing efficiency and maintenance of cell viability. This neuroblastoma cell line was chosen since it contains wild type and could be further used for examination of the cellular effects of knockdown on the DNA damage response. Accell and Non-targeting () control pools as well as Accell sirna and Dharmacon SMARTpool reagents, and two individual sirnas targeting mrna were delivered at 1 µm concentration in Accell sirna Delivery Media. Target mrna knockdown and cell viability were assessed at 72 hours post-transfection (Figure 2). Efficient target mrna knockdown (> 75%) with no detrimental effect on cell viability was observed for all targeting sirna. silencing by Accell sirnas increases the survival of IMR-32 neuroblastoma cells after camptothecin treatment The function of is to keep the cell from progressing through the cell cycle if there is DNA damage by either holding the cell at a checkpoint until repairs can be made and/or causing the cell to enter apoptosis if the damage cannot be repaired 1,2. DNA damaging drugs, such as camptothecin, trigger rapid and extensive apoptosis in chemosensitive human neuroblastoma cell lines. Inactivation of, either by the human papillomavirus type 16 E6 protein or by a dominantnegative mutant (R175H), protects cell lines with wild type from drug-triggered apoptosis 3. In order to demonstrate that knockdown of by Accell sirna would lead to similar protective effects, IMR-32 cells were incubated with Accell sirnas against or control sirna pools (, ) for 72 hours and assayed for cell viability upon treatment with different camptothecin doses for the last 24 hours (Figure 3). The Accell SMARTpool and two individual Accell sirnas targeting caused a striking rescue from camptothecin-induced cell death, as seen in the phase contrast cell images (Figure 3A), and an observed 3-fold increase in cell survival (Figure 3B). Normalized relative target expression and cell viability (%) 1 Target mrna levels pool sirna 1 sirna 2 Figure 2. Efficient target mrna knockdown in IMR- 32 by Accell sirna. 1 µm Accell sirna control pools ( or ) and SMARTpool sirna or individual sirna duplexes against ( pool, sirna 1, and sirna 2) were delivered in IMR-32 neuroblastoma cells in. Knockdown of and and cell viability were assessed at 72 hours posttransfection. Experiments were performed in biological triplicate for all samples. is normally maintained at low levels by continuous ubiquitination and subsequent degradation by the 26S proteasome. However, when the cell is stressed, ubiquitination is suppressed and accumulates in the nucleus, where it is activated and stabilized4.once activated, functions as a transcription factor to turn on or off various genes that affect cell cycle progression and repair (such as p21 Waf1 /Cip1, GAD45α, σ) and/or apoptosis (such as Bax, Apaf1, Casp-9; Figure 1) 5. A. Control () No drug 4 µm camptothecin Thermo Scientific HCS Reagent Kits for multiplex analysis of and p21 expression was used to monitor the effects of down-regulating by Accell sirna. The reduction of the signal and the induction on one of its downstream targets, p21 upon camptothecin treatment was quantified. pool IMR-32 cells were found to be a challenging cell line for HCA due to their loose attachment to the tissue culture plates, so gentle fixing was necessary. This low adherence was further exacerbated by the response of IMR-32 cells to the Accell sirna delivery conditions. contains no serum, as high levels of serum are known to interfere with Accell sirna efficacy. However, the addition of up to 3% serum to is suggested for Accell sirna application in cases where either the cell line or the assay is sensitive to prolonged serum-free conditions. Upon examination of IMR-32 cells under these modified Accell sirna application conditions, it was found that the addition of 2% serum did not affect the target mrna knockdown (Figure 4), but greatly improved cell adherence and fixation steps. The effects of knockdown by Accell sirna on the camptothecin-induced and p21 protein levels were examined next. Treatment with 4 µm camptothecin for hours was found to be the optimal concentration and time for induction of the and p21 protein for the HCA assay in IMR-32 cells (data not shown). 1 No drug 4 µm 1 µm Camptothecin dose Accell Medium pool sirna 1 sirna 2.25 µm IMR-32 cells were incubated with Accell control sirna pools (, ), Accell SMARTpool, or two individual sirnas against. At 52 hours post-delivery the cells were either treated with 4 µm camptothecin for hours or left untreated. At 72 hours cells were fixed and stained with the Thermo Scientific Cellomics multiplexed and p21 detection kit. Cells were analyzed with the Target Detection BioApplication software module. Mean fluorescent intensities of or p21 staining in the nucleus were measured and quantified (Figure 5A). The results show that knockdown of in IMR-32 cells inhibited the and p21 induction following camptothecin treatment. Figure 3. Knockdown of increases the survival of IMR-32 after camptothecin treatment. 1 µm Accell sirna control pools ( or ) and SMARTpool reagent or individual sirnas targeting were delivered to IMR-32 neuroblastoma cells in Accell Delivery Media. At 48 hours post-transfection cells were treated with the indicated doses of camptothecin for 24 hours. was assessed at 72 hours post-transfection by phase contrast cell images (A) or resazurin assay (B).

3 In order to better visualize the effect of knockdown on camptothecindependent induction of and p21, 5 cells treated with either Accell pool or Accell SMARTpool sirna reagent were selected and their nuclear intensity (Hoechst stain) was plotted versus the intensity for p21 stain or stain (Figure 5B). As expected, knockdown of by Accell sirna resulted in a significant decrease of and p21 staining intensities in the cell population, indicating that the cells do not enter cell cycle arrest upon the camptothecin treatment as they do when is not silenced. Thus, we demonstrated that knockdown of by Accell sirna in IMR-32 neuroblastoma cell line results in inhibition of the dependent DNA damage response upon camptothecin treatment. Delivery optimization in primary Cortical Neurons Primary neuronal cultures are widely employed as a highly relevant cellular model system for the study of neurodegenerative diseases. Neurons are post-mitotic, differentiated cells that are cultured under specific conditions. Standard methods applied for modification of target expression by overexpression or by RNAi are often not efficient enough to obtain robust data employing common biochemical assays. The successful application of Accell sirna for delivery into primary neurons would provide great opportunities for RNAi use in target discovery and validation in the field of neuroscience. For this reason, Accell sirna delivery was tested and optimized in primary rat cortical neurons. Primary cortical neurons require specific culturing conditions and are extremely sensitive to medium changes. Therefore a series of experiments was performed in order to assess the effect of Accell delivery conditions on neuronal viability. Primary cortical rat neurons 6 harvested from 18-day embryos (E18) cultured 4 days in vitro (DIV) were incubated either in complete Neurobasal medium, Accell Delivery Media, with B27 supplements or a 5:5 mix of and complete Neurobasal medium. After 48 hours, neuronal viability was assessed by MTT assay (Figure 6). A decrease of cell viability was observed in both with and without B27 supplements relative to Neurobasal medium alone. However, the survival of cells in the 5:5 mixture of and complete Neurobasal medium was not dramatically affected and was further explored for supporting Accell sirna delivery in primary cortical neurons. To determine the optimal medium conditions for the assay, primary E18 rat cortical neurons at 4 DIV were incubated with 1 µm Accell sirna in either Accell (AM) medium alone, complete Neurobasal (NB) medium alone, or different ratios of Accell and Neurobasal Media. Following 48 hours incubation, neuronal viability was assessed by visual inspection (Figure 7A-D) and by MTT assay (Figure 7E). When the primary cortical neurons were incubated in the a substantial decrease in cell viability was observed (Figure 7A, 7E). Cell morphology was not significantly affected by the different ratios of medium compared to Neurobasal medium alone (Figure 7B-D). A decrease in neuronal viability correlated with the quantity of Accell medium present in the delivery mix. Improved survival was obtained when no more than 5% of Accell medium was used in the delivery mix. In parallel, the effect of the different medium conditions on Accell sirna ability to knockdown target mrna was examined (Figure 7F). The expression of mrna was significantly decreased in Relative target expression (%) -2% FBS A. p21 induction (nuclear intensities) 15 5 p21 induction induction (nuclear intensities) 7 induction pool sirna 1 sirna 2 Figure 4. Efficient target mrna knockdown in IMR-32 by Accell sirna delivered in Accell Delivery Media supplemented with low quantity of serum. 1 µm Accell sirnas control pools ( or ) and SMARTpool sirna reagent or individual sirna duplexes against were delivered in IMR-32 neuroblastoma cells in or in with 2% FBS. Knockdown of and was assessed at 72 hours post-transfection. p21 intensity pool sirna 1 sirna 2 pool sirna 1 sirna 2 No treatment 4 µm camptothecin No treatment 4 µm camptothecin pool 5 15 Nuclear Intensity intensity pool pool sirna 1 sirna 2 pool sirna 1 sirna Nuclear Intensity Figure 5. HCA shows reduction in and p21 following camptothecin treatment when is silenced. At 48 hours after sirna delivery, cells were treated with 4 µm camptothecin for hours or were left untreated. At 72 hours after sirna delivery, cells were fixed and stained with the Cellomics Multiplexed and p21 Detection Kit. A. Mean intensities of the nuclear p21 and staining for different sirna treatments. Shows the nuclear Hoechst versus p21 or staining for each individual cell in the population (5 cells) for and sirnas during camptothecin treatment.

4 NB NB+AM AM+B27 AM Figure 6. Testing media compatibility for Accell sirna delivery in primary neurons. Primary E18 rat cortical neurons at 4 DIV were treated for 48 hours with different medium conditions: complete Neurobasal medium (NB), (AM), Accell Delivery Media with B27 supplements (AM + B27) or a 5:5 mix of Accell and Neurobasal medium (NB +AM). MTT assay was performed to assess neuronal viability. A. E. C. F. D. H expression (%) % % 1% 25% 5% % 1% 25% 5% % Figure 7. Testing cell viability and target mrna knockdown in primary neurons upon Accell sirna delivery in different medium conditions. Primary E18 rat cortical neurons at 4 DIV were treated for 48 hours with 1 µm Non-targeting Control () or Accell sirna in different proportions of Accell Delivery Media and complete Neurobasal medium. Phase contrast images of cells treated with sirna in: A., a 5:5 mix of Accell and Neurobasal medium, C. a 25:75 mix of Accell and Neurobasal media, D. complete Neurobasal medium (NB) alone. E. was also assessed by MTT assay, and F. sirna silencing was examined by measuring mrna knockdown. all conditions, with the least efficient knockdown (%) in Neurobasal medium and the highest knockdown efficiency (> 9%) obtained in. To strike a balance between optimal cell viability and target silencing, all subsequent Accell sirna delivery experiments used a 5:5 ratio of Accell medium to Neurobasal medium. In order to more closely examine the Accell sirna delivery, neurons that were incubated for 48 hours with Accell Red sirna (1 µm) in the optimal conditions (5:5 mix of AM:NB) were fixed and images were acquired using a confocal microscope. Almost all neurons were positive for Accell Red sirna uptake (red, Figure 8A and B). Detailed analysis showed that sirnas are localized in the cytoplasm of neuronal cell bodies and in neurites (Figure 8C and D). Knockdown of protects primary neurons from toxicity induced by β-amyloid peptide Neuronal apoptosis can be induced by β-amyloid peptides that play a major role in the pathogenesis of Alzheimer s disease. is a known mediator of β-amyloid peptide neurotoxicity 7. Therefore we examine if silencing of expression using Accell sirna would provide a neuroprotective effect in primary cortical neurons from the β-amyloid peptide. Application of Accell sirna resulted in greater than % knockdown of mrna expression (Figure 9A). Different concentrations of β-amyloid peptide were added to the cells after 48 hours of incubation. The MTT assay was performed at both 48 hours (Figure 9B) and 72 hours (Figure 9C) in order to assess neuronal viability. Silencing of the pro-apoptotic gene leads to a significant increase of neuronal survival as compared to sirna. The neuroprotective effect declined with increased concentration and exposure to β-amyloid. The strongest protective effect was observed at 5 µm β-amyloid at both time points. Reporter-based assays have been described for the validation of functional effects of modulated target expression in neurons 8. However, reporter assays represent an indirect measure of neuronal viability. Moreover, increased toxicity associated with transfection makes it difficult to carry out an adequate evaluation of this phenotype. Here we demonstrate that Accell sirna delivery technology enables the use of whole culture, homogeneous biochemical assays for functional target validation in primary cortical neurons. A. C. Relative expression (%) (untreated control=%) ** 5 µm 1 µm 15 µm β-amyloid peptide concentration (untreated control=%) Figure 8. Analysis of sirna cellular uptake. Primary E18 rat cortical neurons at 4 DIV were incubated for 48 hours with DY-547 labeled Accell sirna (1 µm) in a 5:5 mix of Accell and Neurobasal media. Cells were fixed and images were acquired using a confocal microscope (Zeiss LSM 51). A, C. Detailed analysis shows that sirnas (red) are localized in the cytoplasm in neuronal cell bodies and in neurites; blue staining (Hoechst) indicates nuclei. B, D. Phase contrast images of cells. Scale bar 1 µm µm ** # 1 µm 15 µm β-amyloid peptide concentration Figure 9. Silencing of by Accell sirna causes significant increase of the survival of primary cortical neurons following β-amyloid peptide treatment. Primary E18 rat cortical neurons at 4 DIV were treated with 1 µm Accell sirna pool and Accell sirna pool against (in a delivery medium that represent a 5:5 mix of Accell and Neurobasal medium). A. mrna knockdown was assessed at 48 hours post transfection. At 48 hours post-transfection, cells were treated with different concentrations of β-amyloid peptide. MTT assay was performed after 48 hours and C. 72 hours in order to assess neuronal viability. ** p <.1; # p <.5, t-test.

5 Conclusions The refractory nature of neuronal cells to lipid-based transfection reagents can be overcome with the use of Accell sirna reagents, enabling RNAi in these cell types. An optimization strategy is suggested for identification of conditions to maximize cell viability, and target gene silencing, while accounting for assay-specific requirements. Accell sirna delivery technology permits functional target validation in neuroblastoma cell lines as well as primary cortical neurons. Materials and methods Cell culture IMR-32 cells were obtained from ATCC and cultured under recommended medium conditions. Cells were plated on collagen IV-coated 96-well plates for the purpose of improving the fixation of the cells for HCA analysis. Rat cortical neurons were obtained from E18 pups following Banker, Goslin and Brewer s modified protocol 6. The neurons were cultivated in Neurobasal medium with B27 supplements and used for sirna delivery following 4 days in vitro (DIV). Accell sirna delivery in IMR-32 cells IMR-32 cells were plated at, cells per well in a 96-well plate and allowed to adhere overnight. Accell sirnas used in IMR-32 cells include: Accell Non-targeting pool (Cat #D-191-1), Accell Control Pool (Cat #D-193-1) and Accell sirna pool and individual sirnas against human (Accession #NM_546; Cat #E-3329-, A and A ). Accell sirna was added to Accell sirna Delivery Media (Cat #B-5-) for a final concentration of 1 μm. μl of the Accell sirna and media mixture was then added (per well) to the cells after the growth medium had been aspirated. For the purpose of improving the fixation of the cells for the HCA analysis, the was supplemented with 2% FBS. Cells were incubated for 72 hours at 37 ºC and 5% CO2 at which point the plates were either analyzed for target mrna knockdown, assessed for cell viability, or fixed for HCA analysis. Accell sirna delivery in primary rat neurons Neurons at 4 DIV were incubated for 48 hours with 1 µm Accell sirna in medium containing different proportions of in complete Neurobasal medium (%, 5%, 25%, %) as indicated in the figure legends. Accell sirnas used in primary rat neurons include: Accell Control sirna (Cat #D-193-3), Accell Non-targeting sirna #1 (Cat #D-191-1), Accell SMARTpool against rat (Accession #NM_3989; Cat #E---) and Accell Non-targeting pool (Cat #D-191-1). The cells were analyzed for target mrna knockdown at 48 hours although the protocol recommends a 72 hour time point. For the cellular uptake analysis DY547-labeled Accell Control sirna and Accell Non-targeting sirna were used. For the phenotypic assay the neurons at 4 DIV were treated with 1 μm Accell sirna in the 5:5 mix of and complete Neurobasal medium for 48 hours and then treated with different doses of β-amyloid peptide 1-42 (Californian peptides) for an additional hours. After treatment, the cell viability was assessed by MTT. The β-amyloid peptide 1-42 was prepared according to the previously described protocol 9. assays IMR-32 viability was assessed by resazurin assay. Resazurin was added to cells at a final concentration of 25 μg/ml 72 hours post-delivery. Cells were returned to the incubator for 1-3 hours. Plates were analyzed on a Wallac VICTOR 2 (Perkin Elmer Life Sciences) plate reader (Excitation 53 nm, Emission 59 nm and 1 second exposure). Viability of primary neurons was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay as described previously 1. High content screening assay and analysis IMR-32 cells at 72 hours post-transfection treated with or without 4 μm camptothecin for the last hours were fixed with 4% paraformaldehyde and stained for and p21 proteins. The plates were imaged and quantitatively analyzed on the ArrayScan VTI HCS Reader using the Target Detection Bio Application software module. Three replicate wells for each sirna and treatment (-/+ campthotecin) were analyzed. Eight fields in each well were measured (> cells/field). Parameters used in this analysis included Mean Object Average Intensity in Channel 1 and Mean Average Intensity in Channel 2 and Channel 3. The averages and standard deviations were calculated for each treatment. Numeric data generated by the ArrayScan VTI HCS Reader were evaluated with the vhcs Discovery Toolbox. Confocal microscopy For confocal analysis cells were fixed in 4% paraformaldehyde supplemented with Hoechst nuclear dye. Images were acquired using LSM51 confocal microscope (Zeiss). References 1. K.H. Vousden, X. Lu, Live or let die: the cell s response to. Nat. Rev. Cancer. 2, (2). 2. E.S. Helton, X. Chen, modulation of the DNA damage response. J. Cell. Biochem., (7). 3. H. Cui, A. Schroering, Mediates DNA Damaging Drug-induced Apoptosis through a Caspase-9-dependent Pathway in SH-SY5Y Neuroblastoma Cells. Mol. Cancer Ther. 1, (2). 4. M.F. Lavin, N. Gueven, The complexity of stabilization and activation. Cell Death Differ. 13, (6). 5. T. Riley, E. Sontag, Transcriptional control of human -regulated genes. Nat. Rev. Mol. Cell. Biol. 9, (8). 6. K. Goslin, G. Banker, Experimental observations on the development of polarity by hippocampal neurons in culture. J. Cell Biol. 18, (1989). 7. M.P. Fogarty, E.J. Downer, A role for c-jun N-terminal kinase 1 (JNK1), but not JNK2, in the beta-amyloid-mediated stabilization of protein and induction of the apoptotic cascade in cultured cortical neurons. Biochem. J. 371, (3). 8. G. Pollio, R. Roncarati, A reporter assay for target validation in primary neuronal cultures. J. Neurosci. Methods. 172, (8). 9. W. Stine, Jr., K.N. Dahlgren, In Vitro Characterization of Conditions for Amyloid-beta Peptide Oligomerization and Fibrillogenesis. J. Biol. Chem. 278, (3). 1. T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65, (1983). If you have any questions, contact t +44 () (UK) or (USA); (USA) f +44 () w /contact-us or dharmacon./service-and-support Horizon Discovery, 8 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom Cellomics is a trademark of Thermo Fisher Scientific All trademarks are the property of Horizon Discovery Company unless otherwise specified. 18 Horizon Discovery Group Company All rights reserved. First published January 15. UK Registered Head Office: Building 8, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom. V2-215