Fig 2: RNase L functions in the nucleus and induces nuclear condensation and DNA cleavage in lung epithelial cells. Immunocytofluorescence of RNase L and DAPI in BEAS-2B and NCI-H157 stimulated by 2–5A at the indicated time points. b Immunocytofluorescence of TUNEL and DAPI in BEAS-2B and NCI-H157 stimulated by 2–5A at the indicated time points. c Western blot of RNase L in BEAS-2B interfered with si-RNase L or not. d Immunocytofluorescence of RNase L, DAPI, and Fibrillarin in BEAS-2B interfered with si-RNase L or not. e Western blot of fibrillarin, Topo I, and hnRNP A1 in the nucleus or cytoplasmic fractions; Lamin B and GAPDH are regarded as the internal reference of n nucleus and cytoplasmic fractions, respectively

Fig 3: MeCP2 upregulates Lhcgr as either a CpG islands binding protein or a G-quadruplex binding protein.A Diagram of the Lhcgr gene promoter. Two CpG islands (CGIs, yellow) were located at −1429 bp ~ −1325 bp (CGI-I) and −99 bp ~ 73 bp (CGI-II), respectively; Two putative G-quadruplex sequences (G4, purple) were located at the CGI-II region from −41 bp to −15 bp (G4-1) and 3 bp to 23 bp (G4-2), respectively. The sequences of the two G4s are shown below. B ChIP-qPCR analysis showing the relative enrichment of CGI-I and CGI-II regions in chromatin from the testis of MeCP2Tg1 and WT mice using the MeCP2 antibodies or control IgG (N = 3 for each group). C Luciferase activities of the empty vector- (CONT) or MeCP2- transfected TM3 cells co-expressing the indicated plasmids (shown in the left margin). N ≥ 3 for each group. D Circular dichroism spectra of the Lhcgr-G4-1, Lhcgr-G4-2, c-Myc (positive control), ss1 (negative control) in 100 mM K+ solution. E ChIP-qPCR analysis showing the relative enrichment of G-quadruplexes (G4) regions in chromatin from WT mice testis using the MeCP2 antibodies, G-quadruplex antibodies, or IgG. N = 3 for each group. F Immunoprecipitation assay of BG4-interacting proteins and MeCP2-interacting proteins from TM3 cells transfected with MeCP2 using a BG4 antibody and MeCP2 antibody. The interaction was confirmed by western blot with anti-MeCP2, anti-hnRNPA1, and anti-Vinculin. HnRNPA1, positive control, Vinculin, negative control. G Luciferase activities of the empty vector- (CONT) or MeCP2-transfected TM3 cells co-expressing the indicated plasmids (shown in the left margin). N ≥ 3 for each group. For B, C and E, F, data are presented as mean ± s.e.m. *p < 0.05; ***p < 0.001; ****p < 0.0001, ####p < 0.0001 and n.s. represents not significant. Statistical analyses were performed with One-way ANOVA.

Fig 4: Inhibition of VCP D2-ATPase domain reverses TDP-43, FUS and SFPQ mislocalization phenotypes in VCP mutant MNs. (A) VCP mutant MNs treated with 1 μM of ML240 immunolabelled with TDP-43 and βIII-tubulin. (B) Cell by cell quantification of the nuclear:cytoplasmic ratio (N:C) shows VCP mutant MNs have a loss in the nuclear:cytoplasmic ratio that is increased above control values upon ML240 treatment. (C) Quantification of TDP-43 in the neurites shows an increased nuclear:neurite ratio (Nu:Ne) upon ML240 treatment. (D) FUS and βIII-tubulin immunolabelling in VCP mutant MNs treated with ML240. (E) Quantification of FUS in the nucleus and cytoplasm identify an increase in the nuclear:cytoplasmic ratio upon ML240 treatment in VCP mutant MNs. (F) Quantification of FUS in the neurites shows an increase of the nuclear:neurite ratio to control values upon ML240 treatment. (G) VCP mutant MNs treated with ML240 and immunolabelled with SFPQ and βIII-tubulin. (H) Treatment of ML240 results in no change in the subcellular distribution of SFPQ when examining the nuclear:cytoplasmic ratio (I) but an increase when examining the nuclear:neurite ratio. (J) Quantification of hnRNPA1 shows no change in the nuclear:cytoplasmic ratio upon ML240 treatment in VCP mutant MNs. (K) Quantification of hnRNPK shows no change in the nuclear:cytoplasmic ratio upon ML240 treatment in VCP mutant MNs. Scale bars = 10 μm. Data are collected from 3 independent experimental repeats from 4 VCP mutant ALS lines analysing approximately the following number of cells; MUT1:7000, MUT2:6000, MUT3:7000, MUT4:6000. Data are normalized to control untreated values for each experimental repeat. Data are shown as violin plots with the data points representing the mean for each cell line. Data are plotted by well in graphs B, E, H, J and K and by field of view in graphs C, F and I. Graphs B, E, H and K; P-value is calculated from an unpaired t-test, for graphs C, F, I and J; P-value is calculated from a Mann–Whitney test.

Fig 5: SRSF1 promotes bladder cancer tumorigenesis in part via regulating CD46-CYT2 levels(A) EJ-1 cells were infected with lentivirus expressing several indicated shRNAs to establish stably expressing cell lines. Semiquantitative RT-PCR analysis was performed to detect the CD46 exon 13 alternative splicing. The ratio for 13-/13+ is listed below the panel. (B) CD46 exon 13 splicing was measured by RT-PCR in EJ-1 cells stably expressing FLAG-Cherry, FLAG-SRSF1, or FLAG-hnRNPA1. (C) Relative expression of SRSF1 mRNA expression levels were evaluated by real-time PCR in 27 paired case specimens. Expression levels of SRSF1 were normalized to that of GAPDH. A bar value <1 indicates that SRSF1 is decreased in tumors. A bar value >1 indicates that SRSF10 is increased in tumors. (D) The positive correlation between the CD46 13-/13+ ratio and expression levels of SRSF1 was observed in bladder cancer samples. Relationships between these two variables were determined by Pearson’s correlation coefficients. The correlation was analyzed using GraphPad Prism 5 software. (E) A CCK-8 assay was utilized to quantify cell viability at each time point. Data are plotted as the mean ± SD of three independent experiments and were analyzed by two-way ANOVA. ***p < 0.001. (F) (1) Representative images of cell culture plates following staining for colony formation of the indicated cell lines. (2) The number of colonies was quantified. The data represent mean ± SD and were analyzed by an unpaired two-tailed Student’s t test (n = 3). *p < 0.05. n.s., not significant. (G) (1) Migration assay for the indicated cell lines. (2) Number of migrated cells was quantified in five random images from each treatment group. The data represent mean ± SD and were analyzed by an unpaired two-tailed Student’s t test (n = 5). **p < 0.01, ***p < 0.001.