Fig 2: Effect of SFPQ overexpression on CFTR expression. Overexpression of SFPQ in F508del-CFTR CF lung epithelial cell line (CFBE41o−) was performed using a vector encoding SFPQ. (A) CFBE41o− cells containing the F508del mutation show low levels of CFTR labeling (green) that is faintly perinuclear and punctate. Overexpression of SFPQ in CFBE41o− causes a large increase in the amount of CFTR labeling (green). Green = CFTR, red = phalloidin, blue = DAPI. (B) Expression levels of CFTR protein was analyzed by immunoblot and CFTR mRNA were measured by RT-qPCR analysis using GAPDH mRNA as an endogenous control. (C) Total and surface expression of CFTR was analyzed using a FAP assay in F508del-CFTR CFBE410− cells transfected with the adenoviral vector encoding SFPQ with GFP-tag or control vector encoding GFP alone. The graph indicates data normalized from 4 independent experiments. (D) F508del-CFTR function was analyzed in CFBE cells by Ussing chamber assays. CFBE cells were cultured at the air–liquid interface (ALI), and after differentiation, they were transfected for 48 h with the adenoviral vector encoding SFPQ with GFP-tag or control vector encoding GFP alone. Forty-eight hours post infection, cells were analyzed for short circuit current. The data are representative of two or more independent experiments.

Fig 3: Derivation and validation of NONO-deficient MEFs. (A) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. (B) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female (gt/+); lane 3, Nono-gene trap male (gt/0). (C) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. (D) NONO, SFPQ and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap (gt) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. (E) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.

Fig 4: Impact of SFPQ on gene expression profiles and signaling pathways in CFBE cells. (A) A volcano plot shows the log2 fold change (x axis) plotted against − log10 (adjusted P value [Padj]) (y axis) of total RNA-sequencing (RNA-seq) data compared between SFPQ overexpressed and control CFBE41o− cells (n = 3 each group). Red circles representing significant upregulated genes, blue circles representing significant downregulated genes and grey color circles representing genes those not meeting the threshold. The statistical criterion for a gene to be considered differentially expressed was an adjusted P value < 0.05, FDR < 0.1, > ± 2 FC. (B) Bar graph shows the differential regulation of canonical signal transduction pathways analyzed by Ingenuity Pathway Analysis (IPA) in the SFPQ overexpressed versus control CFBE41o− cells. The bars indicate the − log (P value) of over-representation, while the orange line indicates the threshold, 2.0. The color of the bars indicates the value of the z-score, as indicated in the legends. (C) Top molecular and cellular functions analyzed in the SFPQ overexpressed versus control CFBE41o− cells.

Fig 5: TDP-43 and other disease-linked RNA-binding proteins form nuclear inclusions in GA-Nes mice. a Neuronal nuclear inclusions of TDP-43 (arrow) are detected by immunofluorescence in hippocampus and frontotemporal cortex in GA-Nes end-stage mice, but not Nes littermate controls. No TDP-43 inclusions are detected in affected or asymptomatic PR-Nes mice, or a 68-week Nes control, even in cells with abundant poly-PR (green) inclusions. Scale bar = 10 µm. b Schematic drawing of the distribution of the rare nuclear TDP-43 aggregates in GA-Nes mice. Aggregates were found in the lateral frontal cortex (at least 100 µm from the midline) and hippocampal CA regions marked with red x. c Regional overview of TDP-43 nuclear aggregates (arrows) in the frontal cortex and hippocampus CA3 region of end-stage GA-Nes mice, scale bar = 100 µm. d Nuclear TDP-43 aggregates (arrows) in end-stage GA-Nes mice colocalize with hnRNP H, and weakly with hnRNP A1, but not with Fus, Sfpq or Alyref. No nuclear aggregates of any marker are seen in age-matched Nes mice. Scale bar = 10 µm