LIN28 Overexpression and Knockdown in NPCs

For the LIN28 overexpresssion experiments, we used a commercially available lentivirus expressing human LIN28 (Stemgent, #ST070016) and we used the uninfected cultures as "Control". 1 x 10⁶ transducing units (TU) of the LIN28 lentivirus were used to infect 200,000 WT83 NPCs at passage 4. For the LIN28 knockdown experiments, we used NPCs at passages 2–3 and utilized an shRNA construct targeting human LIN28 in the pLKO.1 vector (TRCN0000102579; Open Biosystems). As the "Control", a pLKO.1 vector containing an shRNA toward GFP was used (Open Biosystems). Both constructs were gifts from Dr. Eugene Yeo used in a previous publication[56 (link)]. The optimal titers of lentiviral supernatants were determined empirically and used to infect WT83 and Q83X NPCs.