Journal: The Journal of Biological Chemistry
Article Title: Abnormal regulation of membrane-less organelles contributes to profilin1-associated ALS
doi: 10.1016/j.jbc.2025.110259
Figure Lengend Snippet: Actin filament agonist CN04 reduces neuronal toxicity caused by PFN1 deficiency . A , HEK 293 cells were transfected with the indicated siRNAs for 48 h, and then the PFN1-depleted cells were treated with 1 μg/ml CN04 for another 12 h. The cells were immunostained with an anti-Coilin (Cajal body) antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. B and C , Quantification of the Cajal body number and size per cell in ( A ). Means ± SD., ∗∗, p < 0.01, p values were determined by one-way ANOVA. D , HEK 293 cells were transfected with the siRNA against PFN1 for 48 h, and then the cells were treated with or without 1 μg/ml CN04 for another 12 h. The cells were harvested for qRT–PCR analysis. The data are presented as means ± SD from three independent experiments. ns, not significantly different, ∗∗, p < 0.01, p values were determined by one-way ANOVA. E-G , mouse primary cortical neurons were transfected with the indicated siRNAs for 48 h, then the neurons were treated with or without 1 μg/ml CN04 for another 12 h. The cells were immunostained with an anti-MAP2 antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. H , quantification of the Cajal body number per cell in ( E - G ). Means ± SD., ∗, p < 0.05, ∗∗, p < 0.01, p values were determined by one-way ANOVA. ( I - J ) Quantification of the neurite length and cell viability in more than 20 neurons ( E - G ). Means ± SD., ∗, p < 0.05, ∗∗, p < 0.01, p values were determined by one-way ANOVA. K , iPSC-derived motor neurons were transfected with the indicated siRNAs for 48 h, then the neurons were treated with or without 1 μg/ml CN04 for another 12 h. The cells were immunostained with an anti-MAP2 antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. L and M , quantification of the number of Cajal bodies and neurite length in more than 20 neurons in ( L ). Means ± SD., ∗, p < 0.05, p values were determined by one-way ANOVA. N , the proposed working model elucidates the role of PFN1 in the context of Cajal body dynamics, Stress granule assembly, and neuronal survival.
Article Snippet: Cytochalasin D (Cyto D) was purchased from MCE (HY-N6682), MG132 was purchased from Calbiochem (474,790), CN04 was purchased from Cytoskeleton (Denver, CO), puromycin was purchased from MCE (HY-B1743).
Techniques: Transfection, Staining, Confocal Microscopy, Quantitative RT-PCR, Derivative Assay
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