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: PFN1 negatively regulates Cajal body formation in non-neuronal and neuronal cells . A , HEK 293 cells were transfected with the indicated siRNAs. After 48 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 ). Mean ± SD., ns, not significantly different; ∗∗, p < 0.01, p values were determined by one-way ANOVA. D , HEK 293 cells were transfected with the indicated siRNAs, and expressing EGFP-Coilin. The cells were visualized by confocal microscopy. Scale bar, 10 μm. E , quantification of the Cajal body number per cell in ( D ). Mean ± SD., ∗∗, p < 0.01, p values were determined by unpaired Student’s t test. F , N2A cells were transfected with the indicated siRNAs. After 48 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. G , quantification of the Cajal body number per cell in ( F ). Mean ± SD., ∗∗, p < 0.01, p values were determined by unpaired Student’s t test. H , schematic diagram depicting the experimental assay to separate MLOs and supernatant in cells. I , HEK 293 cells were transfected with the indicated siRNAs. After 48 h, cell lysates were subjected to droplet co-sedimentation assays and then subjected to immunoblotting using anti-Coilin and GAPDH antibodies (n = 3 biological replicates).

Article Snippet: The following primary antibodies were used: anti-PFN1 antibody (Proteintech, 67,390-1-lg), anti-Coilin antibody (Proteintech, 10967-1-AP), anti-G3BP1 antibody (Proteintech, 13057-2-AP), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-eIF2α (Cell Signaling Technology, 9722), anti-Phospho-eIF2α (Cell Signaling Technology, 9721), anti-puromycin antibody (Abclonal, A23031), and anti-GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Transfection, Staining, Confocal Microscopy, Expressing, Sedimentation, Western Blot

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: PFN1 maintains the liquid-like property of Cajal bodies . A-B , FRAP experiments and live cell imaging of EGFP-Coilin in wild-type and PFN1 depleted cells. Scale bar, 10 μm. C , FRAP recovery curves (quantification data) of experiments in ( A - B ). No statistically significant differences in the size of the Cajal bodies between these two groups. n = 3. D , HEK 293 cells were transfected with the indicated siRNAs and expressed EGFP-Coilin. After 48 h, the cells were treated with 10 μM MG132 for 10 h. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. E , FRAP experiments and live cell imaging of randomly selected EGFP-Coilin in PFN1 knockdown cells treated with DMSO or MG132. Scale bar, 5 μm. F , FRAP recovery curves (quantification data) of experiments in ( E ). n = 4. G , schematic diagram depicting the experimental assay to separate TritonX-100 soluble and insoluble protein fractionation in cells. H and I , HEK 293 cells were transfected with the indicated siRNAs for 48 h, and then the cells were treated with either DMSO or 10 μM MG132 for another 10 h. Cell lysates were subjected to TritonX-100-soluble and insoluble protein fractionation and subjected to immunoblotting using anti-GFP, PFN1, and GAPDH antibodies (n = 3 biological replicates).

Article Snippet: The following primary antibodies were used: anti-PFN1 antibody (Proteintech, 67,390-1-lg), anti-Coilin antibody (Proteintech, 10967-1-AP), anti-G3BP1 antibody (Proteintech, 13057-2-AP), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-eIF2α (Cell Signaling Technology, 9722), anti-Phospho-eIF2α (Cell Signaling Technology, 9721), anti-puromycin antibody (Abclonal, A23031), and anti-GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Live Cell Imaging, Transfection, Staining, Confocal Microscopy, Knockdown, Fractionation, Western Blot

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: PFN1 deficiency causes perturbations in U snRNAs and defects in pre-mRNA splicing . A , HEK 293 cells were transfected with the indicated siRNAs. After 48 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. B , HEK 293 cells were transfected with EGFP-PFN1. After 48 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. C and D , N2A cells and BV2 cells were transfected with the indicated siRNAs. After 48 h, the cells were harvested for qRT–PCR analysis. The data are presented as means ± SD from three independent experiments. ∗∗, p < 0.01, p values were determined by one-way ANOVA. E and F , to evaluate the ratio of spliced versus unspliced mRNA of the indicated gene, N2A cells and BV2 cells were transfected with the indicated siRNAs. After 48 h, the cells were harvested for qRT-PCR analysis. The data are presented as means ± SD from three independent experiments. ∗, p < 0.05, ∗∗, p < 0.01, p values were determined by unpaired Student’s t test.

Article Snippet: The following primary antibodies were used: anti-PFN1 antibody (Proteintech, 67,390-1-lg), anti-Coilin antibody (Proteintech, 10967-1-AP), anti-G3BP1 antibody (Proteintech, 13057-2-AP), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-eIF2α (Cell Signaling Technology, 9722), anti-Phospho-eIF2α (Cell Signaling Technology, 9721), anti-puromycin antibody (Abclonal, A23031), and anti-GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Transfection, Quantitative RT-PCR

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: PFN1 deficiency accelerates Stress granule assembly in response to stress . A , HEK 293 cells were transfected with the indicated siRNAs, and stressed with arsenite (500 μM, 30 min). Subsequently, the cells were fixed, and subjected to immunofluorescence staining using an antibody against G3BP1 (Stress granule), DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. B , quantification of the integrated fluorescence intensity of G3BP1 in the dense phase (condensates) and dilute phase (surrounding nucleoplasm) of cells in (A). C , quantification of the Stress granule size in ( A ). Mean ± SD., ∗∗, p < 0.01, p values were determined by unpaired Student’s t test. D , quantification of the Stress granule number in ( A ). Mean ± SD., ns, not significantly different, p values were determined by unpaired Student’s t test. E and F , HEK 293 cells were transfected with the indicated siRNAs and stressed with arsenite (500 μM, 30 min). Subsequently, the cells were fixed and subjected to immunofluorescence staining using antibodies against G3BP1 and TIA1(Stress granule), DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 5 μm. The fluorescence intensities were quantified on the corresponding lines. G-J , HEK 293 cells were transfected with the indicated siRNAs for 48 h, and then the cells were treated with either DMSO or 500 μM arsenite for 30 min. Cell lysates were subjected to droplet co-sedimentation assays and then subjected to immunoblotting using anti-G3BP1, PFN1, and GAPDH antibodies. The data are presented as means ± SD from three independent biological replicates. ns, not significantly different, ∗∗, p < 0.01, p values were determined by unpaired Student’s t test. K , HEK 293 cells stably expressing EGFP-G3BP1 were transfected with indicated siRNAs. After 48 h, cells were treated with 500 μM arsenite for 30 min to induce Stress granules, and then subjected to FRAP analysis. Scale bars: 5 μm. L , FRAP recovery curves (quantification data) of experiments in ( K ). n = 3. M , HEK 293 cells were transfected with the indicated siRNAs for 48 h, and then the cells were treated with 500 μM arsenite for 30 min. Cell lysates were subjected to TritonX-100-soluble and insoluble protein fractionation and subjected to immunoblotting using anti-G3BP1, PFN1, and GAPDH antibodies (n = 3 biological replicates).

Article Snippet: The following primary antibodies were used: anti-PFN1 antibody (Proteintech, 67,390-1-lg), anti-Coilin antibody (Proteintech, 10967-1-AP), anti-G3BP1 antibody (Proteintech, 13057-2-AP), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-eIF2α (Cell Signaling Technology, 9722), anti-Phospho-eIF2α (Cell Signaling Technology, 9721), anti-puromycin antibody (Abclonal, A23031), and anti-GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Transfection, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Sedimentation, Western Blot, Stable Transfection, Expressing, Fractionation

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: PFN1 modulates Cajal body assembly by regulating actin filament formation . A , HEK 293 cells were treated with 0.1 μg/ml (low dose) or 1 μg/ml (high dose) Cyto D for 1 h. Subsequently, the cells were fixed, and subjected to immunofluorescence staining using an antibody against Coilin (Cajal body), actin filaments were stained using Phalloidin-iFluor 647, and DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. The cartoons below the figures indicate the respective size and number of Cajal body in cells. B and C , quantification of the Cajal body number and size per cell in ( A ). Means ± SD., ns, not significantly different; ∗∗, p < 0.01, p values were determined by one-way ANOVA. D , HEK 293 cells were transfected with the indicated siRNAs. After 48 h, the cells were immunostained with an anti-Coilin (Cajal body) antibody. Actin filaments were stained using Phalloidin-iFluor 647, and DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. E and F , quantification of the Cajal body number and size per cell in ( D ). Means ± SD., ∗∗, p < 0.01, p values were determined by one-way ANOVA. G , HEK 293 cells were treated with 1 μg/ml Cyto D for 1 h and then incubated with 500 μM arsenite for 30 min. Subsequently, the cells were fixed and subjected to immunofluorescence staining using an antibody against G3BP1(Stress granule), actin filaments were stained using Phalloidin-iFluor 647, and DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. H , HEK 293 cells were transfected with the indicated siRNAs. After 48 h, the cells were treated with 500 μm arsenite for 30 min, then the cells were immunostained with an anti-G3BP1 (Stress granule) antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm.

Article Snippet: The following primary antibodies were used: anti-PFN1 antibody (Proteintech, 67,390-1-lg), anti-Coilin antibody (Proteintech, 10967-1-AP), anti-G3BP1 antibody (Proteintech, 13057-2-AP), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-eIF2α (Cell Signaling Technology, 9722), anti-Phospho-eIF2α (Cell Signaling Technology, 9721), anti-puromycin antibody (Abclonal, A23031), and anti-GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Transfection, Incubation

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: ALS-linked PFN1-C71 G mutant impairs the assembly and function of cellular MLOs . A , FRAP experiments and live cell imaging of EGFP-PFN1 C71G cytoplasmic puncta, Scale bar, 10 μm. B , FRAP recovery curves (quantification data) of experiments in ( A ). n = 3. C , HEK 293 cells were transfected with EGFP-alone, EGFP-PFN1 WT , or EGFP-PFN1 C71G . After 24 h, cell lysates were subjected to soluble and insoluble protein fractionation and immunoblotting using anti-GFP and GAPDH antibodies. D , HEK 293 cells were transfected with EGFP-PFN1 WT or EGFP-PFN1 C71G as indicated. After 24 h, the cells were immunostained with an anti-G3BP1 (Stress granule) antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. E , quantification of the percentage of cells with Stress granule in ( D ). F , HEK 293 cells expressing EGFP-PFN1 WT or EGFP-PFN1 C71G were treated with 1 μg/ml puromycin for 15 min, then the cells were subjected to immunoblotting using puromycin and GAPDH antibodies (n = 3 biological replicates). G , HEK293 cells were transfected with EGFP-PFN1 C71G for the indicated time (24 h or 48 h), then the cells were fixed. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. H , HEK293 cells were transfected with EGFP-PFN1 C71G . After 48 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. I , HEK 293 cells were transfected with EGFP-PFN1 WT or EGFP-PFN1 C71G . After 48 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.

Article Snippet: The following primary antibodies were used: anti-PFN1 antibody (Proteintech, 67,390-1-lg), anti-Coilin antibody (Proteintech, 10967-1-AP), anti-G3BP1 antibody (Proteintech, 13057-2-AP), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-eIF2α (Cell Signaling Technology, 9722), anti-Phospho-eIF2α (Cell Signaling Technology, 9721), anti-puromycin antibody (Abclonal, A23031), and anti-GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Mutagenesis, Live Cell Imaging, Transfection, Fractionation, Western Blot, Staining, Confocal Microscopy, Expressing, Quantitative RT-PCR

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: The following primary antibodies were used: anti-PFN1 antibody (Proteintech, 67,390-1-lg), anti-Coilin antibody (Proteintech, 10967-1-AP), anti-G3BP1 antibody (Proteintech, 13057-2-AP), anti-GFP antibody (Santa Cruz Biotechnology, sc-9996), anti-eIF2α (Cell Signaling Technology, 9722), anti-Phospho-eIF2α (Cell Signaling Technology, 9721), anti-puromycin antibody (Abclonal, A23031), and anti-GAPDH antibody (Proteintech, 60004-1-Ig).

Techniques: Transfection, Staining, Confocal Microscopy, Quantitative RT-PCR, Derivative Assay