Fig 2: Expression and subcellular localization of YTHDC1 in oocytes and pre-implantation embryos.(A) YTHDC1 expression in adult mouse tissues. ACTB and TUBB (ß-tubulin) served as loading controls. Heart and skeletal muscle contain little ACTB. (B) Western blot analysis of YTHDC1 in oocytes and pre-implantation embryos. TUBB served as a loading control. Note the lower levels of YTHDC1 in GV-stage oocytes, when normalized to TUBB. (C) Localization of YTHDC1 in oocytes and pre-implantation embryos. DNA was stained with Sytox green. Abbreviations: P5, P12: postnatal days 5, 12; GV, germinal vesicle stage; MII, metaphase II; 1C, 2C, 4C: 1-cell, 2-cell, 4-cell embryos; M/B, morula/blastocyst.

Fig 3: u-Eleanor plays a role in coordinated up-regulation of intragenic Eleanor and ESR1 mRNA to promote the proliferative activity of LTED cells.(a) Overview of a region upstream of the ESR1 locus. The RNA-Seq tracks were aligned with the ChIP-Seq data available in the UCSC genome browser39 (University of California, Santa Cruz, CA, and Supplementary Table 1). Sites amplified by qPCR are shown (a–f). (b) Local expression of u-Eleanor. u-Eleanor was induced at site c in LTED cells, repressed in LTED-RES cells, and de-repressed by ICI 182,780 treatment (ER antagonist, related to Fig. 5e–g). For qRT–PCR, total RNA was pre-treated with DNase I, and the amplification efficiency for each primer set was normalized. The value for site b in MCF7 was set to 1. Values are the means±s.d.; n=3. Corresponding ?Ct values are listed in Supplementary Table 6. (c) RNA Pol II binding to the u-Eleanor region (c) and ESR1 promoter A (f). For ChIP-qPCR, values are the means±s.d.; n=3. P-values were calculated using Student's t-test (**P<0.01). (d) Decreased expression of ESR1 mRNA upon u-Eleanor knockdown by specific siRNAs. Expression levels of ESR1 and ERBB2 mRNAs were measured by qRT–PCR. Values are the means±s.d.; n=3. P-values were calculated using Student's t-test (*P<0.05, **P<0.01). (e) Reduced protein level of ER upon u-Eleanor knockdown. An immunoblot of ER is shown. TUBB served as a loading control. (f) Reduction of Eleanor-containing foci by u-Eleanor knockdown in LTED cells. Independent BAC probes shown in Fig. 1e were used for the FISH analyses. Scale bar, 10 µm. (g) Inhibition of LTED cell proliferation by u-Eleanor knockdown. LTED cells were treated with siRNA targeting u-Eleanor for the indicated periods. Cell numbers are shown as fold changes. Values are the means±s.d.; n=3. P-values were calculated using Student's t-test (*P<0.05, ***P<0.001).