Journal: bioRxiv

Article Title: Novel VCP activator reverses multisystem proteinopathy nuclear proteostasis defects and enhances TDP-43 aggregate clearance

doi: 10.1101/2023.03.15.532082

Figure Lengend Snippet: (A, B) Immunofluorescence confocal images of HeLa cells expressing TDP-4FL treated with 4μM MG132 for 0, 3, or 6 hours stained for myc (TDP-4FL, red) and either ubiquitin (A, green) or VCP (B, green). Scale bar, 10µm. (C) Immunoblots for myc (TDP-4FL) from soluble and insoluble protein fractions from HeLa cells expressing TDP-4FL treated with 4μM MG132 for 0, 3, or 6 hours. GAPDH provided as a loading control. (D-E) Quantification of (D) soluble or (E) insoluble TDP-4FL immunoblots. (n = 4 independent experiment, results are expressed as mean ± SEM over time. One-way ANOVA, ** P < 0.01 for soluble protein, *P < 0.05 for insoluble protein). (F) Anti-myc immunoprecipitation from insoluble protein fractions, immunoblotted for myc (TDP-4FL, red) and ubiquitin (green).

Article Snippet: To test efficacy of UP109 activator, cells expressing myc-TDP-4FL or GFP-TDP-4FL were treated with 4μM of MG132 for 17 hours followed by removal of MG132 and the addition of 30μg/μL of cycloheximide, CHX with DMSO or 5μM or 20μM of UP109 (Enamine Ltd, Kyiv, Ukraine) for 4 hours.

Techniques: Immunofluorescence, Expressing, Staining, Ubiquitin Proteomics, Western Blot, Control, Immunoprecipitation

Journal: bioRxiv

Article Title: Novel VCP activator reverses multisystem proteinopathy nuclear proteostasis defects and enhances TDP-43 aggregate clearance

doi: 10.1101/2023.03.15.532082

Figure Lengend Snippet: (A-B) Analysis of immunofluorescence images to determine the percentage of cells expressing (A) TDP-4FL or (B) eGFP as a percentage of total cell counts after 24 hours of transient TDP-4FL or eGFP transfection in wild-type (WT) HeLa cells or CRISPR-Cas9 knock-in HeLa cells harboring A232E and R155H VCP pathogenic variants. (TDP-4FL, n = 6390 cells counted across all three cell lines over 3 independent experiments; results are expressed as mean percentage values from each independent experiment in addition to overall mean ± SEM. One-way ANOVA, **P < 0.01; Dunnett’s multiple comparison post-hoc, *P < 0.05, **P < 0.01. eGFP, n = 2135 cells counted across all three cell lines over 3 independent experiments; results are expressed as mean percentage values from each independent experiment in addition to overall mean ± SEM. One-way ANOVA, P = 0.459) (C) Representative confocal images of immunofluorescence for TDP-4FL with no MG132 treatment or 4 µM MG132 for 6 hours. Nuclear structures were categorized as either anisosomes or aggregates based on morphology. Scale bar, 10µm. (D) Analysis of immunofluorescence images to determine the proportion of anisosome versus aggregate morphology in WT, A232E, or R155H cells treated with 0, 3, or 6 hours of 4 µM MG132 (n = 7058 cells counted across all three cell lines over 3 independent replicates; results are expressed as mean proportion values from each independent experiment in addition to overall beta ± SE, linear mixed effects model for A232E cells: time P < 0.0001, genotype P = 0.708, time x genotype **P < 0.01; linear mixed effects model for R155H cells: time P < 0.0001, genotype P = 0. 734, time x genotype *P < 0.05). (E) Immunoblots of soluble and insoluble TDP-FL protein in WT, A232E, and R155H cells treated with 0, 3, 6 hours of 4µM MG132. GAPDH shown as a loading control. (F-G) Quantification of immunoblots for (F) soluble and (G) insoluble TDP-FL protein in WT, A232E, and R155H cells treated with 0, 3, 6 hours of 4 µM MG132. (n = 3, results expressed as mean ± SEM over time; two-way ANOVA for soluble protein: time **P < 0.01, genotype P = 0.690, time x genotype P = 0.513; for insoluble protein: time **P < 0.01, genotype *P < 0.05, time x genotype P = 0.210; Dunnett’s post-hoc analysis: *P <0.05).

Article Snippet: To test efficacy of UP109 activator, cells expressing myc-TDP-4FL or GFP-TDP-4FL were treated with 4μM of MG132 for 17 hours followed by removal of MG132 and the addition of 30μg/μL of cycloheximide, CHX with DMSO or 5μM or 20μM of UP109 (Enamine Ltd, Kyiv, Ukraine) for 4 hours.

Techniques: Immunofluorescence, Expressing, Transfection, CRISPR, Knock-In, Comparison, Western Blot, Control

Journal: bioRxiv

Article Title: Novel VCP activator reverses multisystem proteinopathy nuclear proteostasis defects and enhances TDP-43 aggregate clearance

doi: 10.1101/2023.03.15.532082

Figure Lengend Snippet: (A) Schematic of transfection and drug treatments timing to examine the turnover of intranuclear TDP-4FL inclusions. (B) Representative confocal images of immunofluorescence for TDP-4FL in HeLa cells treated with 4µM MG132 for 3 hours (left), versus cells treated with MG132 for 3 hours followed by 30µg/µL CHX for 6 hours (middle) or cells treated with MG132 followed by 30µg/µL CHX + 4µM CB5083 for 6 hours (right). Scale bar, 10µm. (C) Immunoblots for TDP-4FL of soluble and insoluble protein fractions from HeLa cells treated with MG132 versus HeLa cells that recovered with CHX alone or CHX + CB5083. GAPDH shown as a loading control. (D) Quantification of soluble TDP-4FL immunoblots (n = 3 with 2 technical replicates per experiment; data shown as box-and-whisker plots, linear mixed effects model: drug treatment P < 0.0001; Tukey’s post-hoc analysis: ** P <0.01, *** P <0.001). (E) Quantification of insoluble TDP-4FL immunoblots (n = 3 with 2 technical replicates per experiment; data shown as box- and-whisker plots, linear mixed effects model: drug treatment P < 0.0001; Tukey’s post-hoc analysis: ** P <0.01, **** P <0.0001). (F) Representative confocal images of HeLa cells expressing eGFP-TDP4FL treated with 0, 3, or 6 hours of 4µM MG132. Scale bar, 10µm. (G) Wild-type (WT) HeLa cells or CRISPR-Cas9 knock-in HeLa cells harboring (H) A232E and (I) R155H VCP pathogenic variants cells expressing GFP-TDP-4FL were treated with 4µM MG132 for 3 hours, versus cells treated with MG132 for 3 hours followed by 30µg/µL CHX for 6 hours or cells treated with MG132 followed by 30µg/µL CHX + 4µM CB5083 for 6 hours and analyzed by flow cytometry (n = 4 experiments each with 2 technical replicates per condition, integrated fluorescence intensity shown as box-and-whisker plots, linear mixed effects model, drug treatment P < 0.0001; Tukey’s post-hoc analysis, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: To test efficacy of UP109 activator, cells expressing myc-TDP-4FL or GFP-TDP-4FL were treated with 4μM of MG132 for 17 hours followed by removal of MG132 and the addition of 30μg/μL of cycloheximide, CHX with DMSO or 5μM or 20μM of UP109 (Enamine Ltd, Kyiv, Ukraine) for 4 hours.

Techniques: Transfection, Immunofluorescence, Western Blot, Control, Whisker Assay, Expressing, CRISPR, Knock-In, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Novel VCP activator reverses multisystem proteinopathy nuclear proteostasis defects and enhances TDP-43 aggregate clearance

doi: 10.1101/2023.03.15.532082

Figure Lengend Snippet: (A) Schematic of transfection and drug treatments timing to examine the turnover of intranuclear TDP-4FL inclusions. (B) Cells expressing GFP-TDP-4FL were treated with 4µM MG132 followed by 30 µg/µL CHX with or without 5 or 20µM of activator UP109 and analyzed by flow cytometry (n = 4 experiments each with 2 technical replicates per condition, integrated fluorescence intensity shown as box-and-whisker plots, linear mixed effects model: *P < 0.05, **P < 0.01). (C) Representative confocal microscopy images of HeLa cells expressing TDP-4FL after treatment with 4µM MG132 followed by 4 hours of 30µg/µL CHX with or without 5µM UP109. Scale bar, 10µm. (D) Immunoblot for myc (TDP-4FL) of soluble and insoluble protein fractions after treatment with MG132 followed by 4 hours of 30µg/µL CHX with or without 5µM UP109. GAPDH shown as a loading control. (E-F) Quantification of TDP-4FL immunoblots from (E) soluble protein fractions (n = 3 experiments each with 2 technical replicates per condition; data shown as box-and-whisker plots, linear mixed effects model, P = 0.0541) and (F) insoluble protein fractions (n = 3 experiments with 2 technical replicates per condition; data shown as box-and-whisker plots, linear mixed effects model *P < 0.05). (G-H) VCP pathogenic variant harboring (G) A232E and (H) R155H cells were transfected with GFP-TDP-4FL and then treated with MG132 followed by 30µg/µL CHX with or without 5µM of UP109. Integrated fluorescence intensity was measured by flow cytometry (n = 4 experiments each with 2 replicate replicates per condition, data shown as box-and-whisker plots, linear mixed effects model, *P < 0.05).

Article Snippet: To test efficacy of UP109 activator, cells expressing myc-TDP-4FL or GFP-TDP-4FL were treated with 4μM of MG132 for 17 hours followed by removal of MG132 and the addition of 30μg/μL of cycloheximide, CHX with DMSO or 5μM or 20μM of UP109 (Enamine Ltd, Kyiv, Ukraine) for 4 hours.

Techniques: Transfection, Expressing, Flow Cytometry, Fluorescence, Whisker Assay, Confocal Microscopy, Western Blot, Control, Variant Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Prolyl-isomerase Pin1 drives platinum resistance by regulating Notch3 stability and function in ovarian cancer

doi: 10.1186/s13046-026-03658-x

Figure Lengend Snippet: GSK3β phosphorylates Notch3 ICD and induces its proteasomal degradation. a Illustration of the RE/AC domain of N3ICD showing GSK3β consensus motifs (bold: primed residues; red: putative residues) (upper panel) and their sequence alignment across species (lower panel). b MALDI ToF spectra for peptide mass fingerprinting of FLAGN3ICD-wt in the absence (A) or presence of HAGSK3β, alone (B) or with CHIR99021 (2 h) (C), zoomed into the area approximately 3312 m/z corresponding to the 2024LLDQPSGPRSPSGPHGLGPLLCPPGAFLPGLK2055 peptide. Each box contains the MS/MS spectrum of the 3312 m/z signal and the identified phosphorylated residue via Biotools software. c Table showing the different tryptic peptides with the corresponding identified phosphorylated residues (red) detected by an Orbitrap Fusion Tribrid mass spectrometer. nd = not detected. d MALDI ToF spectra for peptide mass fingerprinting of FLAGN3ICD-S2033A alone (A, C) or with HAGSK3β (B, D), zoomed into the area approximately 982 m/z corresponding to the 2024LLDQPSGPR2032 peptide (A, B) or approximately 2253 m/z corresponding to 2033APSGPHGLGPLLCPPGAFLPGLK2055 peptide (C, D). In a-d , numbering refers to UniProtKB entry Q61982 . e-i. HEK293T cells. e Immunoblotting analyses of FLAGN3ICD-wt in the presence of increasing amounts of HAGSK3β. f , g .Immunoblotting analyses of FLAGN3ICD-wt in the presence of the highest dose of HAGSK3β plasmid followed by MG132 (4 h) ( f ) or CHIR99021 (2 h) ( g ). In g , the cells were analysed over a time course with cycloheximide (CHX) (0–2–4 h). h , i . Immunoblotting analyses of FLAGN3ICD-wt and non phosphorylable mutants in the presence or absence of HAGSK3β ( h ) or after a time course with CHX (0-2-4 h) following the overexpression of HAGSK3β (i) (upper panels). Densitometric analyses of β-actin-normalized N3 levels are shown as the mean value of three independent experiments ± SD (lower panels). In i , the results are expressed as percentages with respect to time 0. The difference between FLAGN3ICD-wt and each non phosphorylatable mutant in the presence of HAGSK3β ( h ) or at each time point ( i ) is shown. Statistical significance was computed via one-way ANOVA followed by Sidak’s multiple comparisons test. ns = not significant P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001¸**** P ≤ 0.0001. Anti-β-actin antibody was used as a loading control. In i , * indicates a-specific bands

Article Snippet: Where indicated, the cells were treated with MG132 (50μM for 4 h; #C2211-5MG; Sigma‒Aldrich), cycloheximide (CHX) (10 μg/ml for 0-2-4–6 h; C4859-1ML; Sigma‒Aldrich), Carboplatin (CBDCA) (C2538-100MG - Sigma-Aldrich), Cisplatin (CDDP) (#P4394 - Sigma-Aldrich), CHIR99021 (5μM for 2 h; SML1046-5MG; Sigma‒Aldrich), doxycycline hyclate (1 μg/mL for 48 h; D9891; Sigma‒Aldrich), and Paclitaxel (#33069-62-4 – Selleckchem, Houston, TX, USA) according to the manufacturers’ instructions.

Techniques: Sequencing, Peptide Mass Fingerprinting, Tandem Mass Spectroscopy, Residue, Software, Mass Spectrometry, Western Blot, Plasmid Preparation, Over Expression, Mutagenesis, Control