p21 Cell cycle arrest / genetic repair

Application Note Silencing of expression with Thermo Scientific Dharmacon Accell sirna causes inhibition of the DNA damage response in IMR-32 neuroblastoma cells and protects primary cortical neurons from β-amyloid toxicity Zaklina Strezoska 1 and Tamara Seredenina 2. 1 Thermo Fisher Scientific, Lafayette, CO, USA. 2 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 highly 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 diseaserelevant genes. Thermo Scientific Dharmacon Accell sirna enables 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 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 p21 Stress Cell survival 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 (p21waf1/cip1, GAD45α, 14-3-3α) and/or apoptosis (e.g. Bax, Apaf1, Casp-9). 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. 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 DNA damaging agents (e.g. camptothecin) neurotoxins (e.g. amyloid-β) UV, hypoxia, etc GAD45α 14-3-3σ Bax Apaf-1 Casp -9 Cell cycle arrest / genetic repair P Mdm2 Apoptosis used for examination of the cellular effects of knockdown on the DNA damage response. Accell and Non-targeting () control pools as well as an Thermo Scientific Dharmacon Accell sirna SMARTpool and two individual sirnas targeting mrna were delivered at 1 µm concentration in Accell 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 of 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

Normalized relative target expression and cell viability (%) 14 12 1 8 6 4 2 Figure 3. Knockdown of increases the survival of IMR-32 after camptothecin treatment. 1 µm Accell sirna control pools ( or ) and SMARTpool sirna reagent or individual sirnas targeting were delivered to IMR-32 neuroblastoma cells in Accell delivery media. At 48 hours posttransfection cells were treated with the indicated doses of camptothecin for 24 hours. Cell viability was assessed at 72 hrs post-transfection by phase contrast cell images (A) or resazurin assay (B). A Control () B Normalized cell viability (%) 14 12 1 Target mrna levels Cell viability Figure 2. Efficient target mrna knockdown in IMR-32 by Accell sirna. 1 µm Accell sirnas control pools ( or ) and SMARTpool sirna reagent or individual sirna duplexes against (,, and ) were delivered in IMR-32 neuroblastoma cells in Accell delivery media. Knockdown of and and cell viability were assessed at 72 hrs post-transfection. Experiments were performed in biological triplicate for all samples. 8 6 4 2 No drug No drug 4 µm Camptothecin dose 4 µm camptothecin 1 µm Accell Media.25 µm damage by either holding the cell at a checkpoint until repairs can be made and/or by causing the cell to enter apoptosis if the damage cannot be repaired (reviewed in (Vousden and Lu, 22; Helton and Chen, 27). 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 dominant-negative mutant (R175H), protects cell lines with wild type from drug-triggered apoptosis (Cui et al., 22). 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). High content analysis for the induction of and its downstream target p21 upon camptothecin treatment in IMR-32 cells 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 stabilized (reviewed in Lavin and Gueven, 26). Once activated, functions as a transcription factor to turn on or off various genes that affect cell cycle progression and repair (e.g. p21waf1/cip1, GAD45α, 14-3-3σ) and/or apoptosis (e.g. Bax, Apaf1, Casp-9; Figure 1) (reviewed in Riley et al., 28). The Thermo Scientific Cellomics 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. 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. Accell delivery media 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 Accell delivery media 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 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 Accell delivery media or in Accell delivery media with 2% FBS. Knockdown of and was assessed at 72 hrs posttransfection. Relative target expression (%) 12 1 8 6 4 2 and p21 protein levels were examined next. Treatment with 4 µm camptothecin for 18-22 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). 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 2 hours or left untreated. At 72 hours cells were fixed and stained with the 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. In order to better visualize the effect of knockdown on camptothecin-dependent 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 Accell delivery media Accell delivery media-2% FBS 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 media 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 (Goslin and Banker, 1989) harvested from 18-day embryos (E18) cultured 4 days in vitro (DIV) were incubated either in complete Neurobasal media, Accell delivery media, Accell delivery media with B27 supplements or a 5:5 mix of Accell delivery media and complete Neurobasal

A 2 p21 induction 7 induction P21 induction (nuclear intensities) 15 1 5 P53 induction (nuclear intensities) 6 5 4 3 2 1 B p21 intensity 7 6 5 4 3 2 1 No treatment 4 µm camptothecin No treatment 4 µm camptothecin P53pool intensity 25 2 15 1 5 P53pool Figure 5. HCA shows reduction in and p21 following camptothecin treatment when is silenced. At 48 hrs after sirna delivery, cells were treated with 4 µm camptothecin for 2 hrs or were left untreated. At 72 hrs 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. (B) Shows the nuclear Hoechst versus p21 or staining for each individual cell in the population (5 cells) for and sirnas during camptothecin treatment. Normalized cell viability (%) 12 1 8 6 4 2 5 1 15 2 NB NB+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 media conditions: complete Neurobasal media (NB), Accell delivery media (AM), Accell delivery media with B27 supplements (AM + B27) or a 5:5 mix of Accell delivery media and Neurobasal media (NB +AM). MTT assay was performed to assess neuronal viability. AM+B27 Nuclear Intensity AM media. After 48 hours, neuronal viability was assessed by MTT assay (Figure 6). A decrease of cell viability was observed in Accell delivery media both with and without B27 supplements relative to Neurobasal media alone. However, the survival of cells in the 5:5 mixture of Accell delivery media and complete Neurobasal media was not dramatically affected and was further explored for supporting Accell sirna delivery in primary cortical neurons. To determine the optimal media conditions for the assay, primary E18 rat cortical neurons at 4 DIV were incubated with 1 µm Accell sirna in either Accell (AM) media alone, complete Neurobasal (NB) media alone, or different ratios of Accell and Neurobasal media. Following 48 hours 5 1 15 2 Nuclear Intensity 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 Accell delivery media a substantial decrease in cell viability was observed (Figure 7A, 7E). Cell morphology was not significantly affected by the different ratios of media compared to NB media alone (Figure 7B-D). A decrease in neuronal viability correlated with the quantity of Accell media present in the delivery mix. Improved survival was obtained when no more than 5% of Accell media was used in the delivery mix. In parallel, the effect of the different media conditions on Accell sirna ability to knockdown target mrna was examined (Figure 7F). The expression of mrna

E 1 8 6 4 1% 5% 25% 1% 2 % Normalized cell viability (%) 12 %Accell Delivery Media F 1 8 6 4 2 1% 5% 25% 1% % H expression (%) 12 % Accell Delivery Media Figure 7. Testing cell viability and target mrna knockdown in primary neurons upon Accell sirna delivery in different media conditions. Primary E18 rat cortical neurons at 4 DIV were treated for 48 hrs with 1 µm Non- targeting control () or Accell sirna in different proportions of Accell delivery media and complete Neurobasal media. Phase contrast images of cells treated with sirna in: Accell delivery media (A), a 5:5 mix of Accell and Neurobasal media (B), a 25:75 mix of Accell and Neurobasal media (C) or complete Neurobasal (NB) media alone (D). Cell viability was also assessed by MTT assay (E) and sirna silencing was examined by measuring mrna knockdown (F). Figure 8. Analysis of sirna cellular uptake. Primary E18 rat cortical neurons at 4 DIV were incubated for 48 hrs with DY-547 labeled Accell sirna (1 µm) in a 5:5 mix of Accell delivery media and Neurobasal media. Cells were fixed and images were acquired using a confocal microscope (Zeiss LSM 51). Detailed analysis shows that sirnas (red) are localized in the cytoplasm in neuronal cell bodies and in neurites (C, D). Blue staining (Hoechst) indicates nuclei. Scale bar 1 µm. was significantly decreased in all conditions, with the least efficient knockdown (8%) in Neurobasal media and the highest knockdown efficiency (> 9%) obtained in Accell delivery media. To strike a balance between optimal cell viability and target silencing, all subsequent Accell sirna delivery experiments used a 5:5 ratio of Accell delivery media to Neurobasal media. 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 induces 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 (Fogarty et al., 23). 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 6% 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

A. B. C. Relative expression (%) Cell viability (untreated control=1%) Cell viability (untreated control=1%) 12 1 8 6 4 2 7 6 5 4 3 2 1 6 5 4 3 2 1 5 µm ** ** 1 µm 15 µm 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 media that represent a 5:5 mix of Accell delivery media and Neurobasal media). mrna knockdown was assessed at 48 hours post transfection (A). At 48 hours posttransfection, cells were treated with different concentrations of β-amyloid peptide. MTT assay was performed after 48 hours (B) and 72 hours (C) in order to assess neuronal viability. ** p <.1; # p <.5, t-test. functional effects of modulated target expression in neurons (Pollio et al., 28). 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 # β-amyloid peptide concentration 5 µm 1 µm 15 µm β-amyloid peptide concentration target validation in primary cortical neurons. Conclusions The refractory nature of neuronal cells to lipid-based transfection reagents may be overcome with the use of Accell sirna, 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 media 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 (Goslin and Banker, 1989). The neurons were cultivated in Neurobasal media 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 2, 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 (D-191-1), Accell Control Pool (D-193-1) and Accell sirna pool and individual sirnas against human (NM_546) (E-3329-, A-3329-22 and A-3329-22). Accell sirna was added to Accell delivery media (Cat # B-5-1, www. dharmacon.com) for a final concentration of 1 μm. 1 μl of the Accell sirna and media mixture was then added (per well) to the cells after the growth media had been aspirated. For the purpose of improving the fixation of the cells for the HCA analysis, the Accell delivery media was supplemented with 2% FBS. Cells were incubated for 72 hours at 37 ºC and 5% CO 2 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 media containing different proportions of Accell delivery media in complete Neurobasal media (1%, 5%, 25%, %) as indicated in the figure legends. Accell sirnas used in primary rat neurons include: Accell Control sirna (D-193-3), Accell Non-targeting sirna #1 (D-191-1), Accell SMARTpool against rat (NM_3989) (E-86--2) and Accell Non-targeting Pool (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 Accell delivery media and complete Neurobasal media for 48 hours and then treated with different doses of β-amyloid peptide 1-42 (Californian peptides) for an additional 48-72 hours. After treatment, the cell viability was assessed by MTT. The β-amyloid peptide 1-42 was prepared according to the protocol described in (Stine et al., 23). Assessment of target mrna knockdown Target mrna knockdown in IMR-32 cells was determined by RT-qPCR using Thermo Scientific Verso SYBR Green 2-Step Master Mix (Cat# AB-4112/B, www.abgene.com). and knockdown in primary neurons was determined using iq SYBR Green Supermix (Biorad). To calculate mrna expression levels the CT method was used for IMR-32 cells and the

standard curve method was used for primary neurons. The housekeeping gene, beta-actin was used to normalize gene expression. Expression levels were further normalized to Non-targeting control sirna wells. Experiments were performed in biological triplicate for all samples. Cell viability 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 to 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 (Mosmann, 1983). High content screening assay and analysis IMR-32 cells at 72 hours posttransfection treated with or without 4 μm camptothecin for the last 2 hours were fixed with 4% paraformaldehyde and stained for and p21 proteins using the (product #8462, www.piercenet.com) according to the protocol. The plates were imaged and quantitatively analyzed on the ArrayScan VTI HCS Reader using the Target Detection BioApplication software module. Three replicate wells for each sirna and treatment (-/+ campthotecin) were analyzed. Eight fields in each well were measured (>1 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 Cui, H., Schroering, A., and Ding, H.-F. (22). Mediates DNA Damaging Drug-induced Apoptosis through a Caspase-9-dependent Pathway in SH-SY5Y Neuroblastoma Cells. Mol Cancer Ther 1, 679-686. Fogarty, M. P., Downer, E. J., and Campbell, V. (23). A role for c-jun N-terminal kinase 1 (JNK1), but not JNK2, in the betaamyloid-mediated stabilization of protein and induction of the apoptotic cascade in cultured cortical neurons. Biochem J 371, 789-798. Goslin, K., and Banker, G. (1989). Experimental observations on the development of polarity by hippocampal neurons in culture. J Cell Biol 18, 157-1516. Helton, E. S., and Chen, X. (27). modulation of the DNA damage response. J Cell Biochem 1, 883-896. Lavin, M. F., and Gueven, N. (26). The complexity of stabilization and activation. Cell Death Differ 13, 941-95. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63. Pollio, G., Roncarati, R., Seredenina, T., Terstappen, G. C., and Caricasole, A. (28). A reporter assay for target validation in primary neuronal cultures. J Neurosci Methods 172, 34-37. Riley, T., Sontag, E., Chen, P., and Levine, A. (28). Transcriptional control of human -regulated genes. Nat Rev Mol Cell Biol 9, 42-412. Stine, W. B., Jr., Dahlgren, K. N., Krafft, G. A., and LaDu, M. J. (23). In Vitro Characterization of Conditions for Amyloid-beta Peptide Oligomerization and Fibrillogenesis. J Biol Chem 278, 11612-11622. Vousden, K. H., and Lu, X. (22). Live or let die: the cell s response to. Nat Rev Cancer 2, 594-64. Contact Information For technical questions regarding the use of sirna reagents, please contact Dharmacon Products Technical Support at: In America/Asia 8-235-988 33-64-9499 dharmacon.lab@thermofisher.com In Europe/Israel 8 73724648 32-53-85-71-84 perbio.eurotech@thermofisher.com In Other Countries Please contact your appropriate distributor as listed on www. thermo.com/dharmacondistributors Copyright 29 Thermo Fisher Scientific, Inc. All Rights Reserved. The product(s) described herein ( Products ) are protected by patents, pending patents and other intellectual property owned or licensed by Dharmacon Inc. as set forth in Dharmacon s Terms and Conditions (found at www.thermo.com/dharmacon or included with the Products when sold). By using the Product(s), users accept the Terms and Conditions, which expressly govern all use of the Product(s). The Product(s) are intended solely for research use and not for diagnostic, clinical or therapeutic uses. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Literature Code: 226-9-F-1-U