C., Simpson M. that are important for neuronal development (16, 17). Matricellular proteins are secreted proteins that are closely associated with the extracellular matrix. Rather than being structural components of the extracellular matrix, however, such proteins contribute to cell signaling by acting as BCL2L5 modulators of cell surface receptors. For example, Cyr61 (cysteine-rich 61; CCN1), a member of the CCN (Cyr61/CTGF/NOV) family of matricellular proteins, is a ligand of different integrins (18C20). Through these interactions it regulates cell adhesion, migration, proliferation, survival, apoptosis, differentiation, gene expression, and senescence, depending on the cell type and particular integrin involved (21, 22). Although Cyr61 is mostly known for its roles in various non-neuronal cells (21), it is also expressed in the developing nervous system and neuron-like cells (23, 24). Nonetheless, knowledge about the function of this protein in neurons is very limited. Thus far, the only functional data have come from the work of Kim (24), who showed that knockdown of Cyr61 decreased the etoposide-induced death of immortalized hippocampal progenitor H19-7 cells, suggesting a role of Cyr61 in neuronal cell death. In addition to this observation, the function of Cyr61 in neurons, especially during their development, is unknown, but several factors that induce expression and transcription factors that regulate transcription are known for their contribution to neuronal development and plasticity FXIa-IN-1 (see below). is an immediate early gene, the transcription of which is induced by numerous stimuli, including trophic factors (epidermal growth factor (EGF) and fibroblast growth factor (FGF)) (24, 25), Wnt (26), hypoxia (27), and mechanical stretch (28). The neuronal expression of has not been studied thoroughly, but the activity of the promoter was detected in the developing central nervous system of reporter mouse embryos that carried the -galactosidase (-gal) coding sequence under the promoter (23). It has also been shown that in H19-7 cells, expression can be induced by neurotransmitter glutamate, transcription, activating c-Jun N-terminal kinase (JNK) and serum response factor (SRF). Besides these observations, the regulation of transcription in neurons under more physiological conditions remains rather unexplored. In this work, we first showed that S6K1, the best known mTOR effector, is not sufficient to induce dendritic arbor development. Consequently, we performed short hairpin RNA (shRNA) screening to identify genes that are involved in the regulation of dendritic FXIa-IN-1 arbor shape, the expression of which is potentially regulated by mTOR. In this screen, we identified was obtained by PCR using RAT2 cell cDNA, and the product was cloned into pJET1.2 (Fermentas, Burlington, Canada) according to the manufacturer’s instructions. The pJET-Cyr61 plasmid was next used for the preparation of a template for the cRNA probe with the use of the following primers that contained promoter sequences for T3 or T7 RNA polymerases on their ends: cyr61T7 (5-TAATACGACTCACTATAGGGATGAGCTCCAGCACCATCAAGACGCTCGCTGTCGC-3) and cyr61T3 (5-AATTAACCCTCACTAAAGGGGTCCCTGAACTTGTGGATATCGTTGAACAGACTG-3). pJet-Cyr61 was also used as a PCR template to obtain Cyr61 coding sequence for preparation of -actin-Cyr61-GFP. The obtained PCR product was cloned to EcoRI and SalI sites of -actin-GFP with modified multicloning site. Cell Cultures, Transfection, and Drug and Antibody Treatments The animals used to obtain neurons for tissue cultures and RNA from brain extracts were sacrificed according to the protocols approved by the First Ethical Committee (Warsaw, Poland). Primary hippocampal and cortical cultures were prepared from embryonic day 19 (E19) rat brains and transfected using the Lipofectamine2000 (Invitrogen) or Amaxa nucleofection procedure as described recently (36, 37). For the shRNA library screening, the neurons were transfected with pools of plasmids that encode shRNAs that target a given FXIa-IN-1 gene and GFP-encoding vector in the following proportions: 2:2:2:3 (shRNA#1/#2/#3/GFP) or 3:3:3 (shRNA#1/#2/GFP, in the case that only two shRNAs were used to silence a given mRNA). For insulin-induced dendritic growth, immediately after transfection, the neurons were transferred to a regular culture medium that contained a reduced concentration of B27 (0.2% instead of 2%; Invitrogen). Insulin (400 nm) was added for the first time 4 h after transfection and then every 24 h until cell fixation. To inhibit the 1-integrin receptor, the neurons were treated with anti-CD29 antibody or isotype.