Fig. 4: FUS is required for mOct4P4 lncRNA-mediated silencing of Oct4 in mESCs. | Communications Biology

Fig. 4: FUS is required for mOct4P4 lncRNA-mediated silencing of Oct4 in mESCs.

From: FUS-dependent loading of SUV39H1 to OCT4 pseudogene-lncRNA programs a silencing complex with OCT4 promoter specificity

Fig. 4

a Silver stained protein gel of eluates obtained from mOct4P4-24xMS2 anti-flag RIP experiments. mESCs expressing flag-MS2 and mOct4P4-24xMS2 were used. Indicated bands specifically elute from mOct4P4-24xMS2 lncRNA. Protein identity was determined by mass spectrometry (Supplementary methods). b Anti-flag RIP using mESCs expressing MS2-flag/full length mOct4P4-24xMS2 or 24xMS2 RNA control cells using anti-flag antibody. Agarose gel electrophoresis after quantitative RT-PCR demonstrates the presence of mOct4P4-24xMS2 stem loop RNA (bottom panel). Detection of FUS and MS2-flag proteins by Western blotting (top and middle panel respectively). Bands analyzed by mass spectrometry are indicated as numbers (1–6); complete data on protein identification is available in the provided Supplementary Data 1. c Anti-FUS RIP using MS2-flag mESCs expressing full length mOct4P4-24xMS2 or 24xMS2 RNA control. Presence of FUS and flag-MS2 in eluates was validated by western blotting (top and middle panel respectively). Quantitative RT-PCR followed by agarose gel electrophoresis verified the presence of mOct4P4-24xMS2 in anti-FUS RIP experiments (bottom panel). d FUS and OCT4 western blotting using eluates from mOct4P4-24xMS2 or 24xMS2 mESCs transiently transfected with the indicated siRNAs. ACTIN was used as loading control. Numbers represent OCT4/ACTIN ratio as mean of three independent experiments (24xMS2-CTRL siCTRL was set “100”). e, f ChIP analysis of Oct4 promoter region using an anti-FUS antibody in control or FUS knockdown mESCs (e) or pMEFs (f). Eluates were analyzed by qRT-PCR. g Fus and mOct4P4 expression levels in pMEFs transiently transfected with indicated siRNAs, as determined by qRT-PCR. Expression levels were normalized to Gapdh. h, i qRT- PCR analysis using pMEF cells subjected to siRNA-mediated knockdown of mOct4P4 and Fus. Expression values for Oct4 (h) or self-renewal markers (i) were normalized against Gapdh. Error bars represent standard deviation. Precise p values are indicated. n number of independent experiments carried out.

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