The MEN ε/β transcripts are essential for the integrity of nuclear paraspeckles. (A) Antisense oligonucleotides (ASO) were designed to knock down MEN ε/β or MEN β expression in HeLa cells. Three ASOs (ASO 1, 2, and 3) target both MEN ε and β isoforms, while ASO 4 targets only the MEN β transcript. (Arrows) The positions where each ASO targets the MEN ε/β transcripts. (B) Twenty-four hours after transfection of ASOs into HeLa cells stably expressing EYFP-PSPC1 (also known as PSP1α), an ∼70% knockdown of MEN ε/β (ASO 1, 2, or 3) or 50% knockdown of MEN β (ASO 4) was achieved, as assessed by Q-PCR. Beta-actin was used as a normalization control. The data in the histogram are shown as mean and standard deviation values of three independent experiments. (C) RNA FISH was performed 24 h after HeLa EYFP-PSPC1 cells were transfected with a control ASO or ASOs targeting the MEN ε/β or β transcript, to identify cells in which the RNAs were knocked down. In cells transfected with the control ASO, MEN ε/β transcripts are localized to paraspeckles. Cells transfected with ASO 3 or 4 did not show paraspeckles. (Arrows) Residual paraspeckles in a cell where knockdown of the MEN β transcript was not complete. Scale bar, 10 μm. (D) The portion of paraspeckle-positive cells was reduced to ∼20% by ASO 1, 2, or 3, while control ASO did not appear to influence the integrity of paraspeckles. Treatment with ASO 4 resulted in a loss of paraspeckles, although to a lesser extent. The data in the histogram are shown as mean and standard deviation values of three independent experiments. Approximately 100 cells were counted per experiment. (E) Knockdown of the MEN ε/β transcripts did not result in degradation of the PSPC1 protein. Lamin B1 serves as a loading control.
