RNAi and generation of shRNA transduced MEFs

Lentiviral doxycycline-inducible shRNA constructs for Axin2 (GE Dharmacon, 12006), Tfam (GE Dharmacon, 21780) and RelA (Sigma, TRCN00023583) were purchased for lentivirus preparation. Lentiviral supernatants were collected for 4 days and concentrated using lenti-X concentrator (Clonetech, Cat# 631231). An aliquot of concentrated lentivirus was then used to transduce P0 Nrl–GFP MEFs. For Ascl1 knockdown experiments, control (scramble) shRNA and lentiviral shRNA (a gift from J. Johnson, UT-Southwestern Medical Center; cloned into PLKO.1 (Addgene)) constructs were transduced in MEFs. All lentiviral-transduced cells were selected for 3d in the presence of puromycin (1 μg ml⁻¹). Drug-selected cells were then used for chemical conversion and shRNA induction (Tfam and Axin2) was performed between day 4 and day 8 with doxycycline. Finally, CiPCs were quantified on day 11.