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Fig. 3 A Highly Conserved lincRNA, linc86023, Is Required for the Maintenance of mESC Pluripotency
(A) Schematic of the mouse linc86023 locus on chromosome 12 (UCSC genome version NCBI37/mm9). BC059025 (3,281 bp) and AK045952 (2,876 bp) are alternatively transcribed forms. Blue rectangles represent flanking exons, and blue arrowheads indicate the direction of transcription. Middle panels show ChIP-seq signals of active histone marks H3K4me3 (red) and H3K36me3 (green) in E14 and Bruce4 mESC lines (data from ENCODE/LICR Histone). The bottom profile shows the level of linc86023 sequence conservation in vertebrates.
(B) Northern blot of RNA from CCE mESCs, indicating the linc86023 transcript size (<3000 nt).
(C) RNA FISH for linc86023 in CCE cells.
(D) Linc86023 was found in both nuclear and cytoplasmic fractions. Cellular fractionation was performed in CCE cells followed by RNA isolation, and mRNA levels of XIST, GAPDH, and linc86023 were measured by qRT-PCR. The relative subcellular fraction of each gene was shown.
(E) Alkaline phosphatase staining of CCE mESCs on day 4 following transduction with a control shRNA or 3 independent shRNAs targeting linc86023. Scale bars, 50 µm.
(F) Decreased expression of linc86023 and seven pluripotency genes after knockdown of linc86023 by three shRNAs. qRT-PCR was performed 4 days after transduction. Gene expression was normalized to Actb mRNA levels. Data are the mean ± SD of triplicates.
(G and H) CCE mESC cell proliferation after knockdown (G) and overexpression (H) of linc86023. Cell proliferation (measured as the absorbance at 490 nm) was measured 4 days after shRNA treatment (G) or 5 days after transfection with pcDNA3-linc86023 (H). See also Fig. S3.