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shRNA screen

Manuele Piccolis, Laura M Bond, Martin Kampmann, Pamela Pulimeno, Chandramohan Chitraju, Christina B K Jayson, Laura P Vaites, Sebastian Boland, Zon Weng Lai, Katlyn R Gabriel, Shane D Elliott, Joao A Paulo, J Wade Harper, Jonathan S Weissman, Tobias C Walther, Robert V Farese
Excessive levels of saturated fatty acids are toxic to cells, although the basis for this lipotoxicity remains incompletely understood. Here, we analyzed the transcriptome, lipidome, and genetic interactions of human leukemia cells exposed to palmitate. Palmitate treatment increased saturated glycerolipids, accompanied by a transcriptional stress response, including upregulation of the endoplasmic reticulum (ER) stress response. A comprehensive genome-wide short hairpin RNA (shRNA) screen identified >350 genes modulating lipotoxicity...
February 26, 2019: Molecular Cell
Neeraja M Krishnan, Hiroto Katoh, Vinayak Palve, Manisha Pareek, Reiko Sato, Shumpei Ishikawa, Binay Panda
Tumor suppression by the extracts of Azadirachta indica (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that neem extracts and their associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell lines treated with crude neem leaf extracts, known for their anti-tumorigenic activity...
2019: PeerJ
Chenjian Gu, Shuai Tao, Kongying Hu, Lijun Ming, Mengjun Luo, Huimin Guo, Yu Su, Jing Liu, Youhua Xie
Chronic hepatitis B virus (HBV) infection remains a global public health problem. HBV-encoded X protein (HBx) is a multifunctional regulator that is required to initiate and maintain productive HBV infection, and is involved in HBV-related hepatocellular carcinoma (HCC). Inhibitors that interfere with the functions of HBx could be useful not only for the inhibition of HBV replication but also for the prevention or treatment of HBV-related HCC. To screen molecules that target HBx on a large scale remains a technical challenge due to a lack of sensitive and high-throughput system...
February 28, 2019: Acta Biochimica et Biophysica Sinica
Megan M Tu, Francis Y F Lee, Robert T Jones, Abigail K Kimball, Elizabeth Saravia, Robert F Graziano, Brianne Coleman, Krista Menard, Jun Yan, Erin Michaud, Han Chang, Hany A Abdel-Hafiz, Andrii I Rozhok, Jason E Duex, Neeraj Agarwal, Ana Chauca-Diaz, Linda K Johnson, Terry L Ng, John C Cambier, Eric T Clambey, James C Costello, Alan J Korman, Dan Theodorescu
While a fraction of cancer patients treated with anti-PD-1 show durable therapeutic responses, most remain unresponsive, highlighting the need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies-bladder, breast, colon, sarcoma, and melanoma-we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy...
February 2019: Science Advances
Shu-Hong Zhang, Dong-Xin Liu, Li Wang, Yu-Hua Li, Yan-Hua Wang, Hu Zhang, Zheng-Kang Su, Wen-Gang Fang, Xiao-Xue Qin, De-Shu Shang, Bo Li, Xiao-Ning Han, Wei-Dong Zhao, Yu-Hua Chen
Contactin-associated protein 1 (CASPR1 or CNTNAP1) was recently reported to be expressed in brain microvascular endothelial cells (BMECs), the major component of the blood-brain barrier. To investigate CASPR1's physiological role in BMECs, here we used CASPR1 as a bait in a yeast two-hybrid screen to identify CASPR1-interacting proteins and identified the β3 subunit of Na+ /K+ -ATPase (ATP1B3) as a CASPR1-binding protein. Using recombinant and purified CASPR1, RNAi, GST-pull down, immunofluorescence and -precipitation, and Na+ /K+ -ATPase activity assays, we found that ATP1B3's core proteins, but not its glycosylated forms, interact with CASPR1, which was primarily located in the endoplasmic reticulum of BMECs...
February 21, 2019: Journal of Biological Chemistry
Simona Citro, Alice Bellini, Claudia Miccolo, Lavinia Ghiani, Thomas E Carey, Susanna Chiocca
BACKGROUND: Epidermal growth factor receptor (EGFR) overexpression is associated with the development of head and neck cancer (HNC) and represents one of the main therapeutic targets for this disease. The use of EGFR inhibitors has limited efficacy due to their primary and acquired resistance, partially because of increased epithelial to mesenchymal transition (EMT). The HDAC inhibitor SAHA has been shown to revert EMT in different tumours, including HNC. In this study, we investigated the cooperative role of SAHA and the EGFR tyrosine kinase inhibitor gefitinib in both HPV-positive and HPV-negative HNC cell lines...
February 15, 2019: British Journal of Cancer
Claudia H Huichalaf, Ismael Al-Ramahi, Kyung-Won Park, Stacy D Grunke, Nan Lu, Maria de Haro, Karla El-Zein, Tatiana Gallego-Flores, Alma M Perez, Sung Yun Jung, Juan Botas, Huda Y Zoghbi, Joanna L Jankowsky
An early hallmark of Alzheimer's disease is the accumulation of amyloid-β, inspiring numerous therapeutic strategies targeting this peptide. An alternative approach is to destabilize the amyloid precursor protein (APP) from which Aβ is derived. We interrogated innate pathways governing APP stability using a siRNA screen for modifiers whose own reduction diminished APP in human cell lines and transgenic Drosophila. As proof-of-principle, we validated PKCβ - a known modifier identified by the screen - in an APP transgenic mouse model...
February 12, 2019: Human Molecular Genetics
Coralie Poulard, Hye Na Kim, Mimi Fang, Karina Kruth, Celine Gagnieux, Daniel S Gerke, Deepa Bhojwani, Yong-Mi Kim, Martin Kampmann, Michael R Stallcup, Miles A Pufall
Glucocorticoids (GCs) are used in combination chemotherapies as front-line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although effective, many patients relapse and become resistant to chemotherapy and GCs in particular. Why these patients relapse is not clear. We took a comprehensive, functional genomics approach to identify sources of GC resistance. A genome-wide shRNA screen identified the transcriptional coactivators EHMT2, EHMT1, and CBX3 as important contributors to GC-induced cell death...
February 19, 2019: Proceedings of the National Academy of Sciences of the United States of America
Johannes Berlandi, Amel Chaouch, Nicolas De Jay, Isabel Tegeder, Katharina Thiel, Margret Shirinian, Claudia L Kleinman, Astrid Jeibmann, Paul Lasko, Nada Jabado, Martin Hasselblatt
Background: Recurrent specific mutations in evolutionarily conserved Histone 3 (H3) variants drive pediatric high-grade gliomas (HGG), but little is known about their downstream effects. The aim of this study was to identify genes involved in the detrimental effects of mutant H3.3-K27M, the main genetic driver in lethal midline HGG, in a transgenic Drosophila model. Methods: Mutant and wild-type histone H3.3 expressing flies were generated using a φC31-based integration system...
January 23, 2019: Neuro-oncology
Yoshitaka Sato, Takahiro Watanabe, Chihiro Suzuki, Yuichi Abe, H M Abdullah Al Masud, Tomoki Inagaki, Masahiro Yoshida, Takeshi Suzuki, Fumi Goshima, Jun Adachi, Takeshi Tomonaga, Takayuki Murata, Hiroshi Kimura
Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1 and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral Pre-Initiation Complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. Here, we performed two screens using a kinase inhibitor library, and identified a series of CDK inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through de-stabilization of BDLF4 protein...
January 30, 2019: Journal of Virology
Qi Lei, Chaoji Shi, Jiayi Li, Shengming Xu, Yong Han, Jiang Li, Lei Zhang
BACKGROUND: Yes-associated protein (YAP) is a candidate oncogene in various cancers including oral squamous cell carcinoma (OSCC). Our previous study demonstrated that TNF-alpha could inhibit cell proliferation and invasion by YAP phosphorylation in OSCC. However, the role of YAP in OSCC is not yet clear. The objective of the present study was to elucidate the function of YAP in promoting migration in OSCC and to explore the possible mechanism with a novel YAP inhibitor CA3. METHODS: A total of 68 OSCC patients were enrolled and the expression levels of YAP were investigated in tissue specimens by immunohistochemical staining...
January 29, 2019: Journal of Oral Pathology & Medicine
Mariana Marin, Yulia Kushnareva, Caleb S Mason, Sumit K Chanda, Gregory B Melikyan
The HIV-1 entry pathway into permissive cells has been a subject of debate. Accumulating evidence, including our previous single virus tracking results, suggests that HIV-1 can enter different cell types via endocytosis and CD4/coreceptor-dependent fusion with endosomes. However, recent studies that employed indirect techniques to infer the sites of HIV-1 entry into CD4+ T cells have concluded that endocytosis does not contribute to infection. To assess whether HIV-1 enters these cells via endocytosis, we probed the role of intracellular trafficking in HIV-1 entry/fusion by a targeted shRNA screen in a CD4+ T cell line...
January 25, 2019: Viruses
Kristen E Mengwasser, Richard O Adeyemi, Yumei Leng, Mei Yuk Choi, Connor Clairmont, Alan D D'Andrea, Stephen J Elledge
BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing...
January 23, 2019: Molecular Cell
Michael A Wheeler, Merja Jaronen, Ruxandra Covacu, Stephanie E J Zandee, Giulia Scalisi, Veit Rothhammer, Emily C Tjon, Chun-Cheih Chao, Jessica E Kenison, Manon Blain, Vijayaraghava T S Rao, Patrick Hewson, Andreia Barroso, Cristina Gutiérrez-Vázquez, Alexandre Prat, Jack P Antel, Russ Hauser, Francisco J Quintana
Genome-wide studies have identified genetic variants linked to neurologic diseases. Environmental factors also play important roles, but no methods are available for their comprehensive investigation. We developed an approach that combines genomic data, screens in a novel zebrafish model, computational modeling, perturbation studies, and multiple sclerosis (MS) patient samples to evaluate the effects of environmental exposure on CNS inflammation. We found that the herbicide linuron amplifies astrocyte pro-inflammatory activities by activating signaling via sigma receptor 1, inositol-requiring enzyme-1α (IRE1α), and X-box binding protein 1 (XBP1)...
January 14, 2019: Cell
Julie E McGrath, Louis Panzica, Ryan Ransom, Henry G Withers, Irwin H Gelman
Cancer tumor cell dormancy is a significant clinical problem in breast cancer (BrCa). We used a 3D in vitro model of the endosteal bone niche (EN), consisting of endothelial, bone marrow stromal cells and fetal osteoblasts in a 3D collagen matrix (GELFOAMTM), to identify genes required for dormancy. Human triple-negative MDA-MB-231 BrCa cells, but not the bone-tropic metastatic variant, BoM1833, established dormancy in 3D-EN cultures in a p38-MAPK-dependent manner, whereas both cell types proliferated on 2D plastic or in 3D collagen alone...
January 16, 2019: Molecular Cancer Research: MCR
Ying Ge, Mikkel Bruhn Schuster, Sachin Pundhir, Nicolas Rapin, Frederik Otzen Bagger, Nikos Sidiropoulos, Nadia Hashem, Bo Torben Porse
Cancer sequencing studies have implicated regulators of pre-mRNA splicing as important disease determinants in acute myeloid leukemia (AML), but the underlying mechanisms have remained elusive. We hypothesized that "non-mutated" splicing regulators may also play a role in AML biology and therefore conducted an in vivo shRNA screen in a mouse model of CEBPA mutant AML. This has led to the identification of the splicing regulator RBM25 as a novel tumor suppressor. In multiple human leukemic cell lines, knockdown of RBM25 promotes proliferation and decreases apoptosis...
January 11, 2019: Nature Communications
Chun-Tao Liu, Li Min, Yong-Jun Wang, Peng Li, Yong-Dong Wu, Shu-Tian Zhang
Kinetochore‑associated proteins are critical components of mitotic checkpoints, which are essential for faithful chromosomal segregation and spindle assembly during cell division. Recent advances have demonstrated that kinetochore‑associated proteins are upregulated and serve significant roles in the carcinogenesis of numerous types of cancer. However, the effects of kinetochore‑associated protein 1 (KNTC1) on human cancer, particularly on esophageal squamous cell carcinoma (ESCC), remain unclear. The present study revealed that KNTC1 was highly expressed in ESCC cell lines...
January 3, 2019: International Journal of Oncology
Wenwen Chien, Makoto Sudo, Ling-Wen Ding, Qiao-Yang Sun, Peer Wuensche, Kian Leong Lee, Norimichi Hattori, Manoj Garg, Liang Xu, Yun Zheng, Sigal Gery, Sarawut Wongphayak, Henry Yang, Erkan Baloglu, Sharon Shacham, Michael Kauffman, Seiichi Mori, H Phillip Koeffler
This study is an unbiased genomic screen to obtain functional targets for increased effectiveness of dasatinib in pancreatic cancer. Dasatinib, a multi-targeted tyrosine kinase inhibitor, is used in clinical trials for treatment of pancreatic cancer; however, intrinsic and acquired resistance often occurs. We used a dasatinib-resistant pancreatic cancer cell line SU8686 to screen for synthetic lethality that synergizes with dasatinib using a pooled human shRNA library followed by next generation sequencing...
2018: Journal of Cancer
Dongqing Yan, Anthony D Pomicter, Srinivas Tantravahi, Clinton C Mason, Anna V Senina, Jonathan M Ahmann, Qiang Wang, Hein Than, Ami B Patel, William L Heaton, Anna M Eiring, Phillip M Clair, Kevin C Gantz, Hannah M Redwine, Sabina I Swierczek, Brayden J Halverson, Erkan Baloglu, Sharon Shacham, Jamshid S Khorashad, Todd W Kelley, Mohamed E Salama, Rodney R Miles, Kenneth M Boucher, Josef T Prchal, Thomas O'Hare, Michael W Deininger
Purpose: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR , or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis...
December 18, 2018: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Ugur Eskiocak
Soft agar anchorage-independent growth assays have been commonly used as an indicator of cellular transformation in cell culture. Protocols listed here are optimized to allow for all steps, including plasmid purification, virus production, transduction, and soft agar colony formation, to be performed in 96-well plates. These modifications decrease hands-on time, increase fidelity of the assay, and make it possible to screen 500-1000 short-hairpin RNAs (shRNA) in "one-shRNA-one-well" format in parallel...
2019: Methods in Molecular Biology
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