Fig. 4: Epigenetic and/or transcriptional roadblocks prevent 2C-like conversion in CTCF-depleted ESCCTCF-AID.
a Western blot analysis of CTCF performed in ESCCTCF-AID and NSCCTCF-AID untreated or treated with auxin for four days. Parental ESC were used to show the smaller size of wild-type CTCF compared to tagged CTCF-AID. Tubulin levels are shown as a loading control. b HTI quantification of RFP+ cells in untreated or auxin-treated for four days LTR-RFP reporter parental ESC, NSC, ESCCTCF-AID and NSCCTCF-AID. Center lines indicate mean values. Percentages of RFP+ cells above the threshold are indicated. n = 2000; p-values are shown from two-tailed unpaired t-tests. n.s. = non-significant. Three independent experiments were performed but one representative is shown. c, Box and whisker plot showing the relative fold change (log2) expression of a subset of ten 2 C associated genes (DUX, ZSCAN4, ZFP352, TCSTV3, SP110, TDPOZ1, DUB1, EIF1ad8, PRAMEL7 and MERVLs) in ESC, NSC, ESCCTCF-AID and NSCCTCF-AID untreated or treated with auxin for four days. Reactions were performed by triplicate in two independent experiments. Center line indicates the median, box extends from the 25th to 75th percentiles and whiskers show Min to Max values. p values are shown from two-tailed paired t-tests. n.s. = non-significant. d Representative images from ESCCTCF-AID and NSCCTCF-AID untreated or treated with auxin for four days showing the presence of RFP+ cells. Scale bar, 200 μm. DAPI was used to visualize nuclei. In a–d, one representative experiment is shown but two independent experiments with similar results were performed using two different ESC clones.
