Fig. 7
Ptf1a reprogramming activity depends on Notch-independent interaction with Rbpj. a Schematic depicting the trimeric DNA-binding complex formed among Ptf1a, Rbpj, and an E-protein, as well as the Ptf1aW298A C-terminal mutant that lacks the ability to interact with Rbpj. b Unlike Ptf1a lentivirus-infected MEFs, MEFs transduced with Ptf1aW298A lentiviruses failed to undergo morphological changes by day 6 or form neurospheres by day 9. c Quantification of neurospheres induced by Ptf1a and Ptf1aW298A. MEFs (4 × 104) were seeded into each well of 12-well plates, infected with Ptf1a or Ptf1aW298A viruses, and neurospheres in each well were then counted at day 10 and 14 following virus infection. There was a dramatic decrease of neurospheres in Ptf1aW298A-induced samples at day 10 and 14. Data are presented as mean ± SD (n = 3). Asterisks indicate significance in unpaired two-tailed Student’s t-test: *P < 0.005, **P < 0.0001. d MEFs infected with Ptf1a lentiviruses generated neurospheres at day 8 that were immunoreactive for Sox2, Pax6, and Nestin, whereas MEFs infected with Ptf1aW298A viruses were negative for these NSC markers. Cells were counterstained with nuclear DAPI. e Neurosphere formation by Ptf1a in MEFs infected with lentiviruses expressing Rbpj shRNA or scrambled Rbpj shRNA. f Relative expression levels of Rbpj in MEFs infected with lentiviruses expressing Rbpj shRNA or scrambled Rbpj shRNA as determined by qRT-PCR assay. Data are presented as mean ± SD (n = 6). Asterisk indicates significance in unpaired two-tailed Student’s t-test: *P < 0.0001. g Quantification of neurospheres induced by Ptf1a in the presence of Rbpj shRNA or scrambled Rbpj shRNA. MEFs (4 × 104) were seeded into each well of 12-well plates, infected with Ptf1a lentiviruses and viruses expressing Rbpj shRNA or scrambled Rbpj shRNA. Neurospheres in each well were counted at day 10 following virus infection. Data are presented as mean ± SD (n = 6). Asterisk indicates significance in unpaired two-tailed Student’s t-test: *P < 0.0005. Scale bars, 160 μm (e), 80 μm (b), and 40 μm (d)
