Endogenous intronless mRNAs are specifically detected in NSs. (A) FISH signals with a negative Cntl probe targeting the antisense transcript of the mouse long noncoding RNA Gm14635, SRSF2 IF, and DAPI staining are shown. (B) RT-PCRs to examine the knockdown efficiencies of endogenous intronless mRNAs in cells electronically transfected with ASOs for 48 h. (C–F) FISH signals of endogenous (Endo) intronless mRNAs in HeLa cells were microinjected with a Cntl ASO or ASO targeting the corresponding intronless mRNAs for 10 h. Note that the same exposures were taken for the cells treated with Cntl or transcript-specific ASO. (G and H) RT-qPCRs to examine the levels of the wG mRNA derived from microinjected constructs (G) or endogenous mRNAs (H) in HeLa cells mock-treated or treated with α-amanitin (4 µg/ml) for 6, 12, and 18 h. The graphs show the relative abundance of wG mRNA to DNA or relative abundance of COL1A1, PTB1, and DDX39B pre-mRNAs to 18S rRNA. Data represent the mean ± SEM; n = 3. (I–K) Confocal microscopic images show the distribution of endogenous HSPA1A (I) and ZXDB (J) mRNAs and NSs in HeLa cell mock-treated or treated with α-amanitin (4 µg/ml) for 18 h. The same exposures were taken for the cells treated with Cntl or α-amanitin. The average N/C ratios of each intronless mRNA are shown in the graph (K). The data represent the mean ± SD from three independent experiments; n = 10. Bars, 20 µm. The green and red lines in the graphs demonstrate the mRNA and SRSF2 IF signal intensity, respectively. Statistical analysis was performed using an unpaired t test. ***, P < 0.01.