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: Frontiers in Immunology

Article Title: Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer

doi: 10.3389/fimmu.2017.02004

Figure Lengend Snippet: Metastasis-associated macrophage (MAM) precursor cells (MAMPCs) suppress cytotoxicity of CD8 + T cells through a reactive oxygen species (ROS)-mediated mechanism. (A,B) Effects of myeloid cells on the CD8 + T cell-induced tumor cell apoptosis. Splenic CD8 + T cells from normal C57BL/6 mice were cultured with anti-CD3/CD28 antibodies (effector; E) in the absence or presence of MAMPCs, MAMs, or resident macrophages (RMACs) from the metastatic lung of E0771-LG-injected mice (MAMPC/E, MAM/E, RMAC/E, respectively). The preincubated T cells were then isolated and cultured with E0771-LG cells expressing red fluorescent protein in the nuclei (target; T) at the indicated E:T ratio in the presence of green fluorogenic caspase-3 substrate. After 36 h, the number of apoptotic cancer cells indicated by red/green double positive nuclei was counted. (A) Representative images of cells cultured with the caspase-3 substrate for 36 h (E:T = 4:1). Scale bar; 50 µm, arrowhead; apoptotic cancer cell. (B) Number of apoptotic cancer cells cultured with preactivated CD8 + T cells ( n = 3, two independent experiments). Data are mean ± SEM, * P < 0.01 vs. E + T (4:1), # P < 0.01 vs. MAMPC/E + T. (C) Fold-change of genes encoding checkpoint T cell receptor ligands in MAMPCs and MAMs compared with classical monocytes (C-MOs) determined by microarray analyses in Figure (logFC > 1, FDR < 0.05). Data on expression values are presented as mean ± SEM. Note that the scale is exponential. (D) Mean fluorescence intensity of checkpoint T cell receptor ligands assessed by flow cytometry in C-MOs, MAMPCs, and MAMs ( n = 3, two independent experiments). Blood (for C-MOs) and lung digestion (for MAMPCs and MAMs) were prepared from E0771-LG-injected C57BL/6 mice at 14 days posttumor injection and stained with antibodies for indicated markers or isotype IgG. Data are mean ± SEM, * P < 0.01 vs. IM, # P < 0.01 vs. MAMPC. (E) Effects of checkpoint inhibitors on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and neutralizing antibodies for PD1 or CTLA4, or isotype IgG in the absence (effector; E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio in number of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with IgG in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. IgG. (F) Effects of inhibitors of nitric oxide or ROS production on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and L-NMMA, nor-NOHA, catalase and superoxide dismutase (SOD) (Cat/SOD), or vehicle (–) in the absence (E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with PBS in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. PBS.

Article Snippet: We intraperitoneally injected d -luciferin in PBS (GoldBio, 1.5 mg/100 μL/20 g mouse) into anesthetized E0771-LG:Fl tumor-bearing mice.

Techniques: Cell Culture, Injection, Isolation, Expressing, Microarray, Fluorescence, Flow Cytometry, Staining, Activity Assay