B27 media

U87MG human glioblastoma cells (a gift of Dr. Alonzo Ross, University of Massachusetts Medical School) and HEK293T human embryonic kidney cells (ATCC) were cultured in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic compounds. Cell transfection was performed with Lipofectamine 2000 (Invitrogen) for U87MG cells and calcium phosphate precipitation for HEK293 cells. The culture of primary hippocampal neurons was performed as described (Huang and Richter 2007 (link)) in neurobasal media (Invitrogen) containing B27 supplement (B27 media) and glutamine (1 µg/mL).

Cells were transfected with siRNAs by Dharmafect 1 and typically incubated 72–96 h. Oligonucleotide siRNA sequences were: siFMR1, 5′-GCACUAAGUUGUCUCUGAU-3′; siFXR1, 5′-CGAGCUGAGUGAUUGGUCA-3′; siAUF1, 5′-GAUUGACGCCAGUAAGAAC-3′. siGLD4 was ON-TARGETplus SMARTpool siRNA from Dharmacon. Lentiviral shRNAs targeting CPEB are described elsewhere (Udagawa et al. 2012 (link)). The culture of primary hippocampal neurons was performed as described (Huang and Richter 2007 (link)) in neurobasal media (Invitrogen) containing B27 supplement (B27 media) and glutamine (1 µg/mL). After 14 d, hippocampal neurons were infected with lentivirus expressing shRNA against CPEB and nonspecific shRNA as a control. All oligonucleotide seqences are provided in the Supplemental Information except GLD4 and CPEB1, which were ON-TARGET plus SMARTpool siRNAs from Dharmacon.