Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Antibody panels used for immunohistochemistry on HD tissue microarrays.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemistry, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Immunohistochemical profiling of HTT inclusion body ubiquitination and associated triage protein binding in the HD human middle temporal gyrus. Multiplexed immunohistochemical approaches were used to identify HTT inclusion bodies, ubiquitin species, and triage proteins in neurologically normal and HD human middle temporal gyrus tissue microarray cores. Example images from HD case, HC150, are shown. HTT inclusion body antibodies, EM48 ( A ), EPR ( B ), and MW1 ( C ), were used for labelling together with antibodies for pan-ubiquitin ( D ), K48- and K63-linked polyubiquitination ( E and F ), p62 ( G ), and ubiquilin 2 ( H ), with a Hoechst nuclear counterstain ( I ); scale bars = 20 μm.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemical staining, Ubiquitin Proteomics, Protein Binding, Microarray

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: HTT inclusion bodies are not frequently ubiquitinated, but when ubiquitinated, are predominantly ubiquitinated by K63-linked ubiquitin. Immunohistochemical labelling revealed that EM48, EPR, and/or MW1 HTT inclusion bodies were ubiquitinated by K48- and/or K63-linked ubiquitin ( A ); a representative image of K48- and K63-ubiquitinated HTT inclusion bodies from HD case, HC145, is shown; scale bars = 10 μm. The ubiquitination status of each HTT inclusion body was determined by labelling for pan-, K48-, and K63-linked ubiquitin, where positive labelling was identified if the maximum intensity was above manually determined thresholds. The percentage of EM48 + versus EM48- ( B ), EPR + versus EPR- ( C ), and MW1 + versus MW1- ( D ) HTT inclusion bodies that were ubiquitinated (either pan, K48-, and/or K63-linked) were compared using a Wilcoxon matched-pairs signed rank test. The percentage of ubiquitinated HTT inclusion bodies was determined for each EM48, EPR, and MW1 +/- phenotype per HD case ( E ), and compared between phenotypes using a mixed-effects analysis, with Geisser-Greenhouse correction and Tukey’s multiple comparisons test. The percentage of ubiquitinated HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin was compared using a Wilcoxon matched-pairs signed rank test ( F ). The percentage of ubiquitinated EM48 + versus EM48- ( G ), EPR + versus EPR- ( H ), and MW1 + versus MW1- ( I ) HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin were compared using an ordinary two-way ANOVA with Tukey’s multiple comparisons test. The percentage of EM48, EPR, and MW1 +/- immunophenotypes HTT inclusion bodies identified as being ubiquitinated by K48- or K63-linked chains were compared using an ordinary two-way ANOVA with Sidak’s multiple comparisons test ( J ). Data are presented as truncated violin plots ( n = 20). Statistical significance of differences shown for B-D and F-J: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Statistical significance for E is shown in Supplementary Table 3.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Summary of HTT inclusion body characteristics. Heatmap organised by HTT inclusion body phenotype, with each column representing a single case and each bar coloured according to that case’s value for the characteristic outlined by the row title ( A ). Schematic illustrating the general characteristics of each HTT inclusion body phenotype: (1) EPR + MW1 + inclusion bodies are more frequently located in the nucleus compared to other phenotypes, (2) HTT inclusion bodies that label for more than one epitope-specific antibody are more frequently ubiquitinated, and that ubiquitination occurs more frequently by K63- compared to K48-linked ubiquitin chains, (3) Ubiquitinated HTT inclusion bodies are more frequently tagged by ubiquilin 2 than p62 ( B ). Schematic summarising our hypothesis of HTT inclusion body immunophenotype, ubiquitination, and triage protein tagging with increasing HD severity ( C ); created in BioRender.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics

Journal: Cell reports

Article Title: Mesothelial Cell HIF1α Expression Is Metabolically Downregulated by Metformin to Prevent Oncogenic Tumor-Stromal Crosstalk

doi: 10.1016/j.celrep.2019.11.079

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LY364947 , Tocris Bioscience , 2718.

Techniques: Ex Vivo, Isolation, Microarray, Recombinant, Mutagenesis, Enzyme-linked Immunosorbent Assay, Software, Plasmid Preparation

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. Analysis of USP54 expression from microarray data (GEO accession number GSE27605). High, high Lgr5 or EphB2 receptor expression levels; Med, medium EphB2 receptor expression levels; Low, low Lgr5 or EphB2 receptor expression levels. B. qRT-PCR analysis of USP54 expression in two different colorectal carcinoma xenografts (SP5 and SP9), two-tailed Student's t-test (***, P < 0.001). C. Anchorage-independent growth of control (pLKO.1) and USP54-depleted HCT116 cells (shUSP54.854 and shUSP54.856), two-tailed Student's t-test (**, P < 0.01; ***, P < 0.001). RFU, relative fluorescence units. D. qRT-PCR analysis of USP54 expression in HCT116 cells transduced with control (pLKO.1) or USP54 -specific shRNAs (shUSP54.854 and shUSP54.856). Statistical significance was assessed by two-tailed Student's t-test (***, P < 0.001). E. MTT proliferation analysis of wild-type and USP54-deficient HCT116 cells, Mann Whitney-Wilcoxon test (**, P < 0.01). F. Average area of invasion of HCT116 cells transduced with empty vector (pLKO.1) or USP54 -specific shRNAs (shUSP54.854 and shUSP54.856). Mann Whitney-Wilcoxon test was used to analyze statistical significance (*, P < 0.05; **, P < 0.01). G. Tumor xenograft model performed with subcutaneously injected control and USP54-depleted HCT116 cells. Data are presented as mean ± SEM and statistical significance was assessed by using a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05).

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Expressing, Microarray, Quantitative RT-PCR, Two Tailed Test, Control, Fluorescence, Transduction, MANN-WHITNEY, Plasmid Preparation, Injection

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. , B. Kaplan-Meier survival curves for wild-type and Usp54-deficient males (A) and females (B). C. TaqMan-based qRT-PCR analysis of Usp54 in MEFs and liver tissues from Usp54 +/+ and Usp54 KF/KF mice. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (**, P < 0.01). D. Body weight curves of Usp54 +/+ and Usp54 KF/KF female mice kept on standard diet. E. Body weight curves of Usp54 +/+ and Usp54 KF/KF female mice kept on high-fat diet and a representative image of females of each genotype at the end of the experiment. F. Total weight gain in the same animals. G. Percentage of gonadal and subscapular fat mass with respect to total body weight of the same animals. H. Mean adipocyte area in gonadal and skin fat. I. Representative histological images. Scale bar: 20 μm (gonadal fat) and 200 μm (skin fat). J. Average thickness of the subcutaneous fat deposits for each genotype. Statistical significance was assessed by a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Quantitative RT-PCR, Quantitative Proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. Schematic representation of azoxymethane-induced colon tumor protocol. B. Percentage of animals with the indicated histological alterations. L-Dys, light dysplasia; S-Dys, severe dysplasia; Adeno, adenocarcinomas; Inf. Adeno., infiltrating adenocarcinomas. C. Percentage of infiltrating and mucosal tumors within all carcinomas of each genotype and representative histological images. Scale bar: 500 μm. D. Average number of adenocarcinomas per mouse. E. Length of the colon at the end of the experiment. F. Analysis of USP54 expression from 32 colorectal cancer patient samples, comprising pairs of tumor and matched normal mucosa (GEO accession GDS2947). G. Kaplan-Meier survival plot for 269 patients with intestinal cancer grouped as a function of quantile expressions of USP54 . Statistical significance was assessed by a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; **, P < 0.01).

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Expressing, MANN-WHITNEY

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. TaqMan-based qRT-PCR analysis of Usp54 expression in B16F10 cells transduced with the indicated Usp54-specific shRNA or the empty lentiviral vector (pLKO.1) as a control. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (***, P < 0.001). B. Number of metastases bigger than 200 μm in diameter. Statistical significance was assessed using a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; ***, P < 0.001). C. Representative images of lungs and histological analysis for each condition. Scale bar: 200 μm.

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Quantitative RT-PCR, Expressing, Transduction, shRNA, Plasmid Preparation, Control, Quantitative Proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: Summary of USP54 genetic alterations found in human cancer. ACC, Adrenocortical Carcinoma; Adeno, Adenocarcinoma; CCLE, Cancer Cell Line Encyclopedia; chRCC, Kidney Chromophobe; CS, Carcinosarcoma; DESM, Desmoplastic Melanoma; MPNST, Malignant Peripheral Nerve Sheath Tumor; NEPC, Neuroendocrine Prostate Cancer; PAAC, Acinar Cell Carcinoma of the Pancreas; SC, Small Cell; Sq and Squ, Squamous Cell Carcinoma; ucs, Uterine Carcinosarcoma. Data were obtained from cBioportal ( http://cbioportal.org ).

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques:

Journal: Cell Reports Medicine

Article Title: Arachidonic acid released by PIK3CA mutant tumor cells triggers malignant transformation of colonic epithelium by inducing chromatin remodeling

doi: 10.1016/j.xcrm.2024.101510

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal antibody anti-Ubiquitin , Cell Signaling Technology , Cat# 3936; RRID: AB_331292.

Techniques: Microarray, Recombinant, Cloning, Mutagenesis, Immunohistochemistry, Protein Purification, In Vitro, Sequencing, Methylation

Journal: The Journal of Experimental Medicine

Article Title: Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1 –transformed acute lymphoblastic leukemia cells

doi: 10.1084/jem.20062662

Figure Lengend Snippet: BCR-ABL1–mediated up-regulation of AID involves repression of ID2, a negative regulator of AID. Two Ph + ALL cell lines (BV173 and SUP-B15) were incubated in the presence or absence of 10 μmol/l STI571 for 16 h and subjected to microarray analysis using the Affymetrix U133A 2.0 platform as described in Materials and methods (A). mRNA levels of ID2 were compared with those of AID and its positive regulators E2A and PAX5. As controls, known STI571-inducible genes ( IGKC , RAG1 , RAG2 , and BACH2 ) are shown. (B) The two Ph + ALL cell lines were cultured for 48 h in the presence or absence of STI571, and protein levels of ID2 (top) and AID (bottom) were measured by flow cytometry. (C) To test the functional relevance of BCR-ABL1–mediated down-regulation of ID2 in Ph + ALL cells, the effect of ID2 overexpression on AID mRNA levels was measured in Ph + ALL cells. Therefore, the two Ph + ALL cell lines were stably transduced with a vector encoding only GFP (left) or both GFP and ID2 (right). Overexpression of ID2 was monitored together with mRNA levels of AID. GAPDH mRNA levels were used for normalization of cDNA amounts.

Article Snippet: For analysis of AID and ID2 expression by flow cytometry, antibodies against ID2 (rabbit anti–human ID2 IgG; C-20; Santa Cruz Biotechnology, Inc.) and AID (mouse anti–human AID IgG1; L7E7; Cell Signaling Technology) were used together with secondary antibodies (goat anti–rabbit IgG Cy2 and goat anti–mouse IgG Cy3; Jackson ImmunoResearch Laboratories).

Techniques: Incubation, Microarray, Cell Culture, Flow Cytometry, Functional Assay, Over Expression, Stable Transfection, Transduction, Plasmid Preparation