Fig 1: NSC34 (G4C2)102 cells recapitulate pathological features of C9ORF72-ALS. (A–C, E) NSC34 (G4C2)102 cells were induced with 0.5 µg/ml tetracycline for 5 days. Cells were then stained with a locked nucleic acid (C4G2)3 sense probe (Red), anit-SRSF1 (A), anti-SRSF2 (B) or anti-NCL (C) (all green), and Dapi (Blue). Scale bar = 10 µm. (D) Cerebellar slices from C9ORF72-ALS cases were stained with a locked nucleic acid (C4G2)3 sense probe (Red), anti-NCL (green) and Dapi (Blue). Scale bar = 3 µm. (E) NSC34 sham and NSC34 (G4C2)102 cells were stained for NCL (green) and DAPI (blue). Scale bar = 10 µm. (F) Quantification of NCL area as a percentage of nuclear area in NSC34 sham and (G4C2)102 cells (*p < 0.05; **p < 0.01; two-way ANOVA with Tukey’s multiple comparisons post hoc test; n = 3). (G) Ventral horn from C9ORF72-ALS was stained for NCL and DAPI. Motor neurons (indicated with arrows and nuclei outlined) show disrupted NCL staining, while glial cells do not show disrupted NCL staining. Scale bar = 3 µm.
Fig 2: Reduction of hnRNPA3 increases RNA foci number. Knockdown of hnRNPA3 increases antisense RNA foci in fibroblasts derived from C9orf72 mutation carriers. a Western blotting with anti-hnRNPA3, hnRNPA1, and hnRNPA2B1 antibodies confirms selective and efficient siRNA-mediated knockdown of hnRNPA3. ß-actin serves as a loading control. b Fluorescent in situ hybridization (FISH) of antisense repeat RNA foci in fibroblasts from 3 cases with a C9orf72 repeat extension (C9) and 1 control case (Ct). Arrows indicate Cy3-positive antisense RNA foci in the nucleus. c, d Quantification of RNA foci number and positivity upon siRNA-mediated knockdown of hnRNPA3. N = 126–234 cells from 3 biological replicates in each line. e Western blotting of cell lysates from iPSC-derived human neurons. Western blotting with antibodies to ß-actin and ßIII-tubulin confirmed selective and efficient knockdown of hnRNPA3. f FISH of antisense RNA foci in neurons from control cases (Ct) and C9orf72 carriers (C9). siCt: control siRNA, siA3: hnRNPA3-targeted siRNA. g, h Knockdown of hnRNPA3 increases antisense RNA foci number and positivity in iPSC-derived neurons from C9orf72 mutation carriers. N = 72–102 cells from 3 biological replicates in each line. Data of 6 images in each line were used for analysis. NT non-treatment, siCt control siRNA, siA3 hnRNPA3-targeted siRNA, NCL nucleolin. All graphs are shown as mean ± SEM. *p < 0.05; two tailed paired t test. Scale bar 10 µm
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