kpnb1 egfp  (Addgene inc)

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: N-acetyl-L-leucine (Levacetylleucine) normalizes Transcription Factor EB (TFEB) activity by stereospecific bidirectional modulation

doi: 10.64898/2025.11.30.691375

Figure Lengend Snippet: HeLa cells were transfected with TFEB-EGFP (A-C) or TFEB-mScarlet3 (D,E) and then incubated with NALL at different concentrations or times. Cells were then counter-stained with the NucSpot 488 (D,E) or NucSpot 650 (A-C) to label the nucleus and TFEB translocation quantified by colocalization with NucSpot. (A) Single-cell images of TFEB-EGFP (green) and NucSpot 650 (magenta). (B,C) Concentration-response of HeLa treated for 18 hrs with NALL. Nuclear localization is plotted as either the Pearson’s coefficient (B) or derived as the percentage of cells with nuclear TFEB (C). (D,E) Time course of the effect of 2 mM NALL upon TFEB, either expressed as the Pearson’s coefficient (D) or the percentage of cells with nuclear TFEB (E). The dotted lines (B,D) highlight a correlation coefficient of zero used for thresholding the percentage of nuclear cells. Data are expressed as the mean ± SEM of 179-215 cells, and significance determined by ANOVA test, with significance depicted as * p < 0.05, ** p < 0.01, or *** p < 0.001 compared to respective control, 0 mM (B,C) or t = 0 (D,E).

Article Snippet: Per well, cells were transfected with 100 ng TFEB tagged on its C-terminus with either EGFP (Addgene plasmid # 38119) or mScarlet3 (produced in-house) for 4-6 hrs.

Techniques: Transfection, Incubation, Staining, Translocation Assay, Concentration Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: N-acetyl-L-leucine (Levacetylleucine) normalizes Transcription Factor EB (TFEB) activity by stereospecific bidirectional modulation

doi: 10.64898/2025.11.30.691375

Figure Lengend Snippet: TFEB-EGFP translocation in HeLa cells incubated for 18 hrs with different concentrations of either NALL (black) or L-Leucine (LL, green). Translocation is expressed either as the Pearson’s correlation coefficient with NucSpot 650 (A) or as the percentage of cells with nuclear TFEB (B). Data are expressed as the mean ± SEM of 104-161 cells, and significance determined by nonparametric ANOVA test (A) or unpaired t-test (B), with significance depicted as * p < 0.05, ** p < 0.01, or *** p < 0.001 compared to the vehicle control, i.e. 0 mM. Comparing NALL and LL: #, p <0.05; ###, p < 0.001.

Article Snippet: Per well, cells were transfected with 100 ng TFEB tagged on its C-terminus with either EGFP (Addgene plasmid # 38119) or mScarlet3 (produced in-house) for 4-6 hrs.

Techniques: Translocation Assay, Incubation, Control

Journal: bioRxiv

Article Title: N-acetyl-L-leucine (Levacetylleucine) normalizes Transcription Factor EB (TFEB) activity by stereospecific bidirectional modulation

doi: 10.64898/2025.11.30.691375

Figure Lengend Snippet: TFEB-EGFP translocation in HeLa cells incubated for 18 hrs with different concentrations of each enantiomer: the L-form (NALL, black), D-form (NADL, green) or the racemic mixture of both (orange). Translocation is expressed either as the Pearson’s correlation coefficient with NucSpot 650 (A) or as the percentage of cells with nuclear TFEB (B). Data are expressed as the mean ± SEM of 153-265 cells, and significance determined by nonparametric ANOVA test, with significance denoted by * p < 0.05, ** p < 0.01, or *** p < 0.001 compared to vehicle control, i.e. 0 mM. Comparing NALL and NADL or racemic mixture, using a nonparametric ANOVA test: ##, p < 0.01; ###, p < 0.001.

Article Snippet: Per well, cells were transfected with 100 ng TFEB tagged on its C-terminus with either EGFP (Addgene plasmid # 38119) or mScarlet3 (produced in-house) for 4-6 hrs.

Techniques: Translocation Assay, Incubation, Control