Journal: Neurotherapeutics

Article Title: Small molecule JRMS modulating importin-β1 chaperone activity as a therapeutic strategy reducing TDP-43 pathology

doi: 10.1016/j.neurot.2026.e00834

Figure Lengend Snippet: JRMS reduces TDP-25 aggregates in mouse primary cortical neurons. (a) Neurons treated with up to 30 μM JRMS exhibit no significant cytotoxicity. (b) Immunofluorescence imaging showing reduction of TDP-25 aggregates (green; arrows) in JRMS-treated neurons compared to DMSO control. DAPI (blue) Quantification showing reduced (c) number and (d ) size of aggregates in JRMS-treated neurons compared to DMSO control. (e) Immunoblot of total lysates in neurons transduced with EGFP, EGFP-TDP-25 and EGFP-TDP-43, treated with 15 μM JRMS or DMSO, labelled for KPNB1, EGFP (JL8 antibody), pTDP (409/410) and GAPDH. JRMS reduces phosphorylated TDP-25 (arrow) without affecting the total level of expression of the EGFP-tagged proteins. Quantification shows similar levels of EGFP-TDP-25 (f) , and reduced phosphorylated TDP-25 (g) in JRMS-treated neurons compared to DMSO control. (h) Quantification of KPNB1 levels shows no difference between JRMS-treated and DMSO control. Scale bar = 20 μm; ∼250 neurons across N = 3 biological replicates per treatment; Statistical analysis was performed using two-tailed t -test ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001 .

Article Snippet: The pcDNA3.1 constructs for transfection have been described previously, and include: EGFP, EGFP-TDP-25, EGFP-TDP-35, EGFP-TDP-43ΔNLS (K82A, R83A and K84A), EGFP-TDP-C-spl-272, RFP-TDP-25, 3 × FLAG-mTB-TDP-25 [ , ] and KPNB1-EGFP (Addgene plasmid # 106941).

Techniques: Immunofluorescence, Imaging, Control, Western Blot, Transduction, Expressing, Two Tailed Test

Journal: Neurotherapeutics

Article Title: Small molecule JRMS modulating importin-β1 chaperone activity as a therapeutic strategy reducing TDP-43 pathology

doi: 10.1016/j.neurot.2026.e00834

Figure Lengend Snippet: The effect of JRMS on reducing TDP-25 aggregation is dependent on KPNB1. (a) Immunoblot showing that siRNA knockdown of KPNB1 increases insoluble and phosphorylated EGFP-TDP-25 (pTDP), whereas increased expression of KPNB1-EGFP reduces insoluble and phosphorylated Flag-TDP-25. (b) Quantification of amount of insoluble pTDP, normalized to GAPDH within each fraction. (c) Immunofluorescence image showing expression of KPNB1-EGFP (green) reduces Flag-TDP-25 aggregates (red; arrowhead), quantified in (d) . DAPI (blue). (e) Immunoblot showing effect of JRMS in reducing insoluble, phosphorylated EGFP-TDP-25 is prevented in cells with siRNA knockdown of KPNB1. (f) The quantification of insoluble pTDP faction in DMSO vs JRMS in siScram and siKpnB1 cells. (g) JRMS treatment increases the cytoplasmic localization of KPNB1-EGFP, quantified in (h) . (i) Immunoblot showing that endogenous KPNB1 co-immunoprecipitates with EGFP-TDP-25, with apparent reduced association in conditions of JRMS treatment as quantified in (j) . However, (k) normalization to pTDP shows increased interaction, indicating that JRMS promotes KPNB1 binding to aggregated TDP-25. (l) JRMS treatment does not affect nucleocytoplasmic ratio of endogenous TDP-43 (red), quantified in ( m ) or of the ( n ) NLS-tdTomato-NES NCT reporter (red), quantified in (o) . DAPI (blue). Scale Bar = 10 μm; N = 3 biological replicates; ∼100 cells quantified per biological replicate in C, G, L and N; Statistical analysis was performed using two-tailed t -test for two condition comparison or one-way ANOVA for multi-condition comparison ∗p < 0.05 ∗∗p < 0.01 ∗∗∗p < 0.001 .

Article Snippet: The pcDNA3.1 constructs for transfection have been described previously, and include: EGFP, EGFP-TDP-25, EGFP-TDP-35, EGFP-TDP-43ΔNLS (K82A, R83A and K84A), EGFP-TDP-C-spl-272, RFP-TDP-25, 3 × FLAG-mTB-TDP-25 [ , ] and KPNB1-EGFP (Addgene plasmid # 106941).

Techniques: Western Blot, Knockdown, Expressing, Immunofluorescence, Binding Assay, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Two sequential waves of mRNA translation drive embryonic development

doi: 10.1101/2025.09.18.676998

Figure Lengend Snippet: a Double staining of GFP-Ewsr1b (green) and GFP-Ewsr1b mRNA (red) in embryos injected with GFP-Ewsr1b mRNA carrying Long-3′UTR (Long) or Short-3′UTR (Short) at 4 hpf. Scale bars: 20 µm. b Violin plots showing distances from the nuclear center to signals of GFP-Ewsr1b mRNA carrying Long-3′UTR or Short-3′UTR (means ± SD; n = 80). Similar results were obtained from two independent experiments. **********p < 0.0000000001 (Student’s t -test). c Immunofluorescence of Importin β1 in embryos at 3 hpf. d Immunoblotting of embryos at 3 hpf following IP with control IgG (IgG) or anti-Importin β1 (α-Im β) antibody, and RT-PCR for ewsr1b -3′Long and α-tubulin mRNAs. Similar results were obtained from two independent experiments. e Double staining of Importin β1 (red) and the ewsr1b -3′Long mRNA 3′UTR (green) in embryos at 3 hpf. Left: High-resolution confocal image; right: enlarged views of the boxed area. Similar results were obtained from two independent experiments. f Immunofluorescence of Importin β1 and Ewsr1b in uninjected embryos (Control) or embryos injected with Importazole at 3 hpf. DNA is shown in blue. Enlarged views of the boxed area with or without DNA staining are shown on the right side. Scale bars, 10 µm. g Quantification of average signal intensity in the nucleus per 25 µm 2 . (means ± SD; n = 10). ***********p < 0.00000000001 (Student’s t -test).

Article Snippet: Proteins were separated by SDS-PAGE, transferred onto Immobilon membranes, and probed with primary antibodies; mouse anti-Syncrip antibody (1:1,000, hnRNP Q; Santa Cruz Biotechnology, I8E4; sc-56703), rabbit anti-Syncrip antibody (1:1,000, Proteintech, 14024-1-AP), rabbit anti-Rpl11 antibody (1:1,000, Abcam, ab79352), rabbit anti-Pou5f3 antibody (1:100), mouse anti-GFP antibody (1:1,000, Roche, 11814460001), mouse anti-Ewsr1b antibody (1:100, present study), rabbit anti-Importin β1 antibody (1:1,000, Proteintech, 10077-1-AP), and mouse anti-HuR antibody (1:1000, Santa Cruz Biotechnology; sc-5261).

Techniques: Double Staining, Injection, Immunofluorescence, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Scientific Reports

Article Title: In-frame germline TP53 variant impairs p53 oligomerization and predisposes to cancer

doi: 10.1038/s41598-025-14684-8

Figure Lengend Snippet: Impaired transcriptional activity of the p53 p.E339_F341del isoform. ( A ) Map of the TP53 genetic locus targeted by CRISPR/Cas9. Parental RPE cells were transfected by sgRNA and Cas9, were grown in the presence of nutlin-3 and two clones of RPE-TP53-KO cells were expanded. Genomic DNA was sequenced by NGS. Partial sequence of exon 4 of the TP53 is shown with the target sequence of sgRNA underlined. Note two frameshifting mutations corresponding to the two alleles in RPE-TP53-KO cells. ( B ) Whole cell lysates from parental RPE and RPE-TP53-KO cells incubated or not with nutlin-3 for 12 h were analyzed by immunoblotting. Note induction of p53 and p21 signal after nutlin-3 treatment in parental cells and the absence of p53 and p21 signal in RPE-TP53-KO cells. Staining for importin beta which is an abundant protein involved in nucleocytoplasmic trafficking was used as a loading control . ( C ) Parental RPE and RPE-TP53-KO cells treated with nutlin-3 for 12 h were fixed by PFA, permeabilized by 0.1% TX-100 and analyzed by immunofluorescence microscopy. Representative image is shown. ( D ) Parental RPE, RPE-TP53-KO and RPE-TP53-KO cells stably transfected with wt-p53 (positive control), p53-R248W (negative control) and E339_F341del plasmids were treated with doxycycline and nutlin-3 for 12 h. Whole cell lysates were analyzed by immunoblotting. Staining for importin beta and histone H3 was used as loading controls. ( E ) Parental RPE, RPE-TP53-KO and RPE-TP53-KO cells stably transfected with wt-p53, p53-R248W and E339_F341del plasmids were treated with doxycycline and nutlin-3 for 12 h. After fixation and permeabilisation, cells were probed with p21 and p53 antibodies and analyzed by ScanR microscopy. Mean nuclear intensity of p21 signal was determined in > 300 non-gated RPE and RPE-TP53-KO cells or in the p53-positive RPE-TP53-KO cells rescued by the wild-type or mutant p53. Plotted is the mean ± SD from independent biological replicates (n = 3) normalized to p21 levels in cells expressing the wild type p53. Statistical significance was determined by t-test, ** P < 0.01. ( F ) Parental RPE, RPE-TP53-KO and RPE-TP53-KO cells stably transfected with wt-p53 (positive control), p53-R248W (negative control) and E339_F341del plasmids were treated as in ( E ) and were probed with MDM2 and p53 antibodies. Mean nuclear intensity of MDM2 signal was determined as in ( E ). ( G ) FASAY analysis of the p53-E339_F341del variant transformed into yeast strain yIG397. White colonies (45.3%) contain the functional p53. The fraction of red colonies containing a transcriptionally inactive p53 allele was 54.7%, indicating that the patient is a heterozygote carrying one functional and one transcriptionally inactive p53 allele. Representative image is shown.

Article Snippet: The following antibodies were used: p53 (sc-6243, IF dilution 1:100), p21 (sc-6246, IF dilution 1:100, WB 1:1000), and importin beta (sc-137016) from Santa Cruz; MDM2 (OP46, IF dilution 1:100) from Calbiochem; histone H3 (14269S, WB 1:1000), GAPDH (5174S, WB 1:1000) and p53 (9282S, WB 1:1000) from Cell Signaling Technology; Alexa Fluor-conjugated secondary antibodies (Thermo Scientific).

Techniques: Activity Assay, CRISPR, Transfection, Clone Assay, Sequencing, Incubation, Western Blot, Staining, Control, Immunofluorescence, Microscopy, Stable Transfection, Positive Control, Negative Control, Mutagenesis, Expressing, Variant Assay, Transformation Assay, Functional Assay