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. 2015 Dec 21;10(12):e0145336. doi: 10.1371/journal.pone.0145336

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© 2015 Yang et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Fig 1 — (A) MEFs were infected with increasing dose of FUW-M2rtTA and TetO-FUW-OSKM viruses in two independent infection experiments, and treated with doxycycline for 2 days. The mRNA levels of Set1/Mll complex subunits was determined by RT-qPCR and normalized by Gapdh. Average ± range of values from duplicate assays are plotted. (B) MEFs were infected with increasing dose of FUW-M2rtTA and TetO-FUW-OSKM viruses and treated with doxycycline for 2 days, and the expression of core subunits and global H3K4me3 were examined by western blotting. (C and D) MEFs were infected with virus expressing indicated individual reprogramming factor together with FUW-M2rtTA virus. MEFs infected with only FUW-M2rtTA virus was used as control (Con), and some MEFs were co-infected with TetO-FUW-Oct4 and TetO-FUW-Sox2 viruses (OS), in addition to FUW-M2rtTA. After induction with doxycycline for 2 days, the mRNA (C) and protein (D) levels of indicated Set1/Mll complex subunits were determined by RT-qPCR and normalized by Gapdh (C) and western blotting (D). Average ± SD from 3 independent infections are plotted in (C). (E and F) MEFs were infected with viral mixes that expressed individual Oct4, Sox2, Klf4 (OSK) or Oct4, Sox2, Klf4, and c-Myc (OSKM), in addition to FUW-M2rtTA, and MEFs infected with only FUW-M2rtTA virus was used as control. After induction with doxycycline for 2 days, the mRNA levels (E) of indicated core subunits were determined by RT-qPCR and normalized by Gapdh (E, left) or Actb (E, right). Average ± range of values from 2 independent infections are plotted. The indicated proteins were determined by western blotting (F). (G) Structure of human and mouse Wdr5 genes showing the highly conserved intronic sequences flanking the canonic E box. Identical residuals between human and mouse are in red. (H) Left: P493-6 cells were cultured in the absence (-Tet, Myc on) or presence of (+Tet, Myc off) tetracycline, and used for Myc ChIP followed by qPCR on indicated loci. Middle: MEFs were infected with only FUW-M2rtTA virus (control) or with FUW-M2rtTA and TetO-FUW-OSKM viruses (OSKM), induced with doxycycline, and used for Myc ChIP followed by qPCR on indicated loci. Right: MEFs were infected with FUW-M2rtTA and TetO-FUW-Myc virus (Myc) or FUW-M2rtTA and TetO-FUW-HA-Myc viruses (HA-Myc), induced with doxycycline, and used for HA ChIP followed by qPCR on indicated loci. Wdr5 dst, a Wdr5 downstream region. Cad is a previously established Myc target [63] and used as a positive control. Note that there are E boxes at +10088 and +23063 bp in the Dpy30 gene body. Average ± SD from triplicate assays are plotted, except for Myc ChIP in MEFs, for which Average ± range of values from duplicate assays are plotted. The differences between blue and red bars in all three panels are statistically significant (P<0.05 in 2-tailed Student’s t-test) for all loci except for the “Wdr5 dst” and the “Intergenic” sites.