Fig. 1: Conserved function of hOCT4P3 and mOct4P4 lncRNAs.

a Schematic representation of murine mOct4P4 and human hOCT4P3 pseudogenes. Length of sequence elements and percentage of sequence homology are indicated. Gray boxes, sequences with homology to Oct4/OCT4 5′UTR; gray lines, sequences with homology to Oct4/OCT4 3′UTR. A centrally located, 334-bp spliced fragment is exclusively present in mOct4P4 (29). b Subcellular localization of hOCT4P3 in human Ovarian Cancer cell lines OVCAR-3, SKOV3, TOV-112D, and CAOV3 as determined by quantitative RT-PCR (qRT-PCR). Shown values refer to the percentage of total RNA expression. c Quantitative RT-PCR analysis of hOCT4P3 (left panel), OCT4 and pluripotency marker genes (right panel) in OVCAR-3 cells stably expressing hOCT4P3. Expression levels were normalized against ACTIN. d dCas9-HA-KRAB western blotting analysis (top) and RT PCR analysis (bottom) of Oct4 pseudogene guide RNA (sgOct4P4, sgOCT4P3) in mouse embryonic stem cells (mESCs) (left panel) and OVCAR-3 cells (right panel). ACTIN and Gapdh were used as control. e, f mOct4P4 lncRNA (e) and Oct4 (f) expression in self-renewing mESCs (EB T0) and during 10 days of embryoid body (EB) differentiation (EB D3–D10). Expression levels were normalized to Gapdh. g qRT-PCR analysis of self-renewal marker genes (left panel) or markers of early mESC differentiation (right panel) in dCas9/sgOct4P4 mESCs. Expression values were normalized against gapdh. h Percentage of contractile cardiomyocyte structures in embryoid bodies (EBs) obtained from dCas9 or dCas9/sgOct4P4 cells. i, j Quantitative RT-PCR showing hOCT4P3 lncRNA (i) and OCT4 (j) expression in dCas9 or dCas9/sgOCT4P3 OVCAR-3 cells. Expression values were normalized using ACTIN. k OCT4 expression in knockdown dCas9 and dCas9/sgOCT4P3 OVCAR-3 cells as determined by western blotting. ACTIN was used as control. Numbers represent OCT4/ACTIN ratio (dCAS9 empty was set “100”). l Chromatin immunoprecipitation (ChIP) analysis on the OCT4 promoter region in dCas9 and dCas9/sgOCT4P3 OVCAR-3 cells using H3K9me3 antibodies. Error bars represent standard deviation; Precise p values are indicated; n number of independent experiments carried out.