Pcagig

HepG2 cells (ATCC, HB-8065) were cultured in Eagle’s Minimum Essential Medium (Quality Biological, 112-018-101CS) supplemented with 1x GlutaMAX (ThermoFisher Scientific, 35050061), 10% FBS (Corning, 35-010-CV) and 1% Penicillin-Streptomycin (ThermoFisher Scientific, 15070063). siRNA targeting PTBP1 (Sigma, SASI_Hs01_00216644) or eGFP as negative control (ThermoFisher Scientific, AM4626) were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, L3000-008) following the manufacturer’s protocol. For overexpression of MSI1 and PCBP2, Ultimate ORF expression clones from ThermoFisher Scientific (MSI1 - IOH41182, PCBP2 - IOH4487) were cloned into pCAGIG (Addgene, 11159) and again transfected using Lipofectamine 3000. For experiments involving a combination of plasmid overexpression and siRNA knockdown, plasmids were first transfected at 0 h, siRNA were transfected at 24 h, and cells were processed two days later at 72 h. For FACS isolation, cells were dissociated using TrypLE (ThermoFisher Scientific, 12604013) to form a single-cell suspension and sorted by GFP fluorescence on a BD FACSCalibur in the JHMI Ross Flow Cytometry Core Facility.

The sMarf1 open reading frame (ORF) was amplified from cDNA obtained from mouse cortical neurons using specific primers (Forward: 5′-CCATATTGTCTGGCTATGT-3′, Reverse: 5′-TCCATGTTCAAATGGGAG-3′). Then, cDNA was cloned into a pCR Blunt II TOPO vector (Thermo Fisher Scientific) with the restriction enzyme sites NotI and XhoI. Next, the sMarf1 ORF was inserted into a pCAGIG (Addgene) vector by restriction enzyme digestion using a DNA ligation kit ver. 2.1 (TaKaRa). To add the V5-tag to the C-terminus of sMarf1, the sMarf1 ORF was subcloned into a pcDNA3.1/V5-His TOPO vector (Thermo Fisher Scientific). In parallel, to establish the ΔNYN sMarf1 construct, the sMarf1 ORF was amplified using specific primers (Forward: 5′-CACCATGGGCCACACTCTACTCTAT-3′, Reverse: 5′-TTTAAGCTTGGTTACAGGTGCAAAAG-3′), and the product was cloned into the pcDNA 3.1/V5-His TOPO vector. Finally, to express the proteins in neuronal cells, both reading frames were transferred to a pCAGIG vector containing a V5 epitope tag. For the construction of the sMarf1 shRNA vector, oligos (Sense: 5′-GATCCCCACACCTCACTTGTGCACCATTCAAGAGATGGTGCACAAGTGAGGTGTTT-3′, Anti-sense: 5′-AGCTTAAAAAACACCTCACTTGTGCACCATCTCTTGAATGGTGCACAAGTGAGGTGT-3′) were designed using OligoEngine software. The oligos were annealed and cloned into a pSUPER retro.neo + gfp vector (Addgene).

The pCAGIG plasmid (Addgene plasmid #11159) was inserted with a fragment encoding a nuclear localization signal (NLS) and 3×Flag in the EcoRI site. To make pCAGIG-Prdm16-FL or pCAGIG-Prdm16-PRdeletion, the full-length open reading frame (ORF) or the truncation that lacks the coding sequence for amino acids 2-180 of Prdm16 was PCR amplified from MSCV-Prdm16 (Addgene plasmid #15504) and inserted between the EcoRI and XhoI sites in pCAGIG-NLS-Flag. The VP64 fragment was then inserted to the XhoI site of pCAGIG-Prdm16 to make pCAGIG-FL-VP64.
The plasmids used for making stable cell lines, pCDH-Prdm16 and pCDH-Prdm16-PRdeletion, were generated as follow: The Prdm16 FL ORF, the PR-deletion coding sequences or the NLS-3×Flag was digested from their pCAGIG plasmids and inserted sequentially to the pCDH-CMV-MCS-EF1-Puro plasmid (System Biosciences) between the EcoRI and NotI sites (for Prdm16-FL and Prdm16-PRdeletion) and the XbaI and EcoRI sites (for NLS-3×FLAG).
To clone shFlrt3, annealed oligos were cloned into the HpaI and XhoI sites of the lentiviral vector pLL3.7, which contains a separate CMV promoter that drives the expression of dsRed. Flrt3 shRNAs effectively suppressed co-expressed Flag-FLRT3 protein in HEK293T cells.