Plix403

Neurons (at day 4 of the differentiation process) were seeded at 1x10⁵ cells/well in 6-well plates at 40% confluency and transduced overnight with lentivirus (200 μl at MOI > 5) expressing an NS or FMR1-SF shRNA. Cells were selected with 0.5 μg/ml puromycin for 3 days, and then transduced overnight with a lentivirus (400 μl at MOI 5) expressing an NS or FMR1 shRNA (TRCN0000059762), or expressing empty vector (pLIX_403, Addgene Plasmid #41395) or REST (pLIX-REST, Addgene Plasmid #91896). Cells were harvested for analysis by qRT-PCR or immunoblot at day 16 post-differentiation at 50% confluency. For small molecule FMR1-SF inhibitor treatment, neurons (at day 4 of the differentiation process) were first transduced overnight with a lentivirus (200 μl at MOI > 5) expressing an NS or FMR1 shRNA, or expressing empty vector or REST, selected with 0.5 μg/ml puromycin for 2 days, and then incubated with a small molecule inhibitor (1 μM 5-aza-2′-deoxycytidine, 0.5 μM chaetocin, 5 μM EPZ6438, 5 μM GSK126 or 5 μM PRT4165) for 96 h. Cells were harvested for analysis by qRT-PCR or immunoblot at day 11 post-differentiation at 50% confluency.

Plasmids used here include: pBAD33-V5-DarT (Thermus aquaticus), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa (Thermus aquaticus), pET28a-6xHis-DarG-K80A-1–155aa (Thermus aquaticus), pLIX_403-GFP-DarT-WT (Thermus aquaticus), pLIX_403-GFP-DarT-E160A (Thermus aquaticus), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

MAPT cDNA (NM_005910.6) with a single-mutation (1216C>T) and GFP cDNA were synthesized and inserted into the doxycycline-inducible lentiviral vector PLIX_403 (Addgene #41395). Lentiviral particles were generated by the MD Anderson Functional Genomics Core Facility.