Fig 1: NEAT1, P54nrb, and PSPC1 co-localize with HSV-1 genomic DNA. The biotin-labeled HSV-1 genomic DNA probe (green) incubated with denatured (a) or undenatured (b) HeLa cells nuclei infected with HSV-1(blue) and then incubated with the NEAT1 probe (red), the anti-P54nrb antibody (red) or anti-PSPC1 antibodies (red). The images were captured with a confocal microscope. The intensity plots for the red and green channels were analyzed with ImageJ software. DAPI (blue) was used to stain the nuclei. Scale bars 10 µm
Fig 2: PSPC1 levels are reduced by 2'-F-modified oligonucleotides. (A) Indicated oligonucleotides were transfected into HeLa cells at a final concentration of 30 nM. After 24 h, levels of PSPC1 were evaluated by western. GAPDH served as a loading control. (B) A431 cells were incubated with ISIS404130 by free uptake for 40 h at the indicated concentrations. Levels of P54nrb, PSF and PSF proteins were evaluated by western. GAPDH served as a loading control.
Fig 3: HSV-1 infection redistributes speckles and paraspeckles.a HeLa cells infected with HSV-1 or Mock for 4 h were incubated with anti-SRSF2 antibodies (green) and then incubated with NEAT1 probe (red), anti-PSPC1 antibodies (red), or anti-P54nrb antibodies (red). The images were captured with a confocal microscope. The intensity plots for the red and green channels were analyzed using ImageJ. DAPI (blue) was used to stain the nuclei. Scale bar, 10 μm. b The Pearson’s coefficient and overlap coefficient for each merge channel in (a) were quantified using the JACoP in ImageJ. The data are presented as the mean ± standard deviation (SD) from three independent experiments (*p < 0.01, Student’s t-test). c Schematic diagram showing the primer-amplified regions (black box) in the NEAT1 sequence. d HeLa cells infected with HSV-1 or Mock for 4 h were harvested and subjected to a RIP assay. QRT-PCR was performed to detect the retrieval of NEAT1 by anti-SRSF2 antibodies over the input level. The data are presented as the mean ± SD from three independent experiments (*p < 0.01, Student’s t-test). e HeLa cells infected with HSV-1 or Mock for 4 h were fixed, incubated with DIG-labeled NEAT1-N2 fragment (green), NEAT1-N4 fragment (green), and SRSF2 (red), and subjected to confocal microscopy analysis. The intensity plots for the red and green channels were analyzed using Image J. DAPI (blue) was used to stain the nuclei. Scale bar, 10 μm. f The Pearson’s coefficient and overlap coefficient of each merge channel in (e) were quantified for each merge channel using the JACoP in ImageJ. The data are presented as the mean ± SD from three independent experiments (*p < 0.01, Student’s t-test, NS no significance). g HeLa cells were infected with HSV-1 or Mock for 4 h. The cell lysates were then harvested and subjected to an immunoprecipitation assay with anti-SRSF2 antibodies or anti-IgG antibodies. The retrieval of PSPC1 and P54nrb by endogenous SRSF2 or IgG and the input levels of SRSF2, PSPC1, and P54nrb were measured by western blotting.
Fig 4: HSV-1 infection increases NEAT1 expression and paraspeckle formation. a HeLa and MEF cells were infected with HSV-1 and harvested at the indicated time points. The expression levels of NEAT1 relative to those of ß-actin mRNA were determined with real-time PCR. The data were normalized to the NEAT1 level at 0 h after HSV-1 infection. b HeLa cells were infected with HSV-1 and collected at the indicated time points for western blot to analyze the expression of ICP0, P54nrb, PSPC1, STAT3, pSTAT3 Y705 and ß-actin. c HeLa cells were fixed 4 h after HSV-1 infection and incubated with the NEAT1 probe (red). Fluorescence images were captured with a confocal microscope. Scale bars 10 µm. The number of NEAT1 puncta per cell was analyzed. *p < 0.01
Fig 5: P54nrb preferentially associates with 2'-F-modified oligonucleotides. (A) Oligonucleotides used in this experiment. (B) ASO-binding proteins were isolated from HeLa cell extracts using a biotinylated gapmer 2'-F-PS-ASO (ISIS623496). Proteins were eluted from beads by competition with indicated gapmers. The affinity-selected proteins were analyzed by western blot. Ku70 served as a loading control. (C) Western analysis following ASO-pull down as described in panel (A) indicated that P54nrb has higher affinity for oligonucleotides with the 2'-F modification on the 3' side. Ku70 served as a loading control. (D) Transfection of 2'-F-oligonucleotides reduced P54nrb but not FUS protein levels. HeLa cells were transfected with 2'-F-PS-ASO (ISIS404130) at a final concentration of 30 nM, and protein levels were determined by western analysis. (E) Western analysis of proteins isolated from HeLa cells following treatment of cells with siRNAs targeting P54nrb or PSPC1 mRNA. Cells were transfected with siRNA at 3-nM final concentration and harvested after 36 h.
Supplier Page from Abcam for Anti-PSPC1 antibody