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: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Cell stem cell

Article Title: Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

doi: 10.1016/j.stem.2017.04.009

Figure Lengend Snippet: Figure 7. a-ITGA7 Impairs GBM Tumor Growth and Invasion In Vivo (A) Luciferase activity detected with a Xenogen IVIS 100 small animal in vivo imaging system, 5 days post-sc transplantation of 5 X 105 GSC1-LUC cells in mice either treated with anti-ITGA7 (a-ITGA7) antibody or isotype control (i.p.). (B) Analysis of sc GSC1-LUC tumor growth over time in mice treated either with isotype control or with a-ITGA7 antibody (1.4A12). Shown is the average photon count and SEM of n = 6 mice/group measured by Xenogen IVIS 100small animal in vivo imaging system (***p< 0.001, two-tailed Student’s t test). See also FigureS7. (C) Analyses of the tumor volumes 5 months after ceasing the anti-ITGA7 treatment. Shown is the size of the sc tumors of n = 6 mice/group measured by calliper (**p < 0.01, two-tailed Student’s t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PE Mouse monoclonal anti EphA2 (clone 371805) R&D Cat#FAB3035P; RRID:AB_11128496 APC Mouse monoclonal anti CD44 BD Biosciences Cat# 560890 Mouse monoclonal anti CD44 (clone DF1485) DAKO Cat#M7082; RRID:AB_2076596 Mouse monoclonal anti ITGA7 Miltenyi Bioteck Cat#130-103-774 Mouse monoclonal anti murine ITGA7 (clone 6A11) Acris Cat#AM20012AF-N; Please See Table S3 for Reverse Phase Protein Microarray Antibodies used N/A N/A Biological Samples GSC (Glioblastoma Stem Cells) (Marziali et al., 2016; Ricci-Vitiani et al., 2008) N/A Chemicals, Peptides, and Recombinant Proteins EGF Peprotech Cat#100-15 bFGF Peprotech Cat#100-18B Apotransferrin Sigma Cat#T2252 Putrescine Sigma Cat#P5780 Sodium Selenite Sigma Cat#S5261 Bovine Serum Albumin Sigma Cat#A2153 Progesterone Sigma Cat#P8783 Insulin Sigma Cat#I3536 HAT Supplement 50X Thermo Scientific Cat#21060017 HT Supplement 100X Thermo Scientific Cat#11067030 BM Condimed H1 Hybridoma Suppl.

Techniques: In Vivo, Luciferase, Activity Assay, In Vivo Imaging, Transplantation Assay, Control, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: NOX2 enhances FADDosome-induced gastric epithelial mitochondrial fission and dysfunction in PHG. (A) Two-dimensional hierarchical clustering results for the enzyme NADPH oxidase (NOX) family were presented for the comparison between PHG patients and healthy volunteers (n = 3 per group). The fold changes and P values of the indicated mRNAs in PHG tissues relative to those in normal (Uninvolved) tissues from the microarray experiment were also presented. (B) NOX1, NOX2 and NOX4 IHC staining (brown) showed that NOX2, but not NOX1 or NOX4, was upregulated in the gastric mucosal samples of PHG patients, and the NOX1, NOX2 and NOX4 areas were also analyzed. n = 6 per group. * P < 0.05. (C) Representative images of IHC staining (brown) for NOX1, NOX2 and NOX4 in mouse models revealed that Nec-1 (an inhibitor of RIPK1) repressed NOX2 expression in mice with PVL. n = 6 per group. (D) The NOX1, NOX2 and NOX4 areas detected by IHC staining in (C) were presented. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (E) The colocalization of TOMM20 (red) and NOX2 (green) determined by confocal imaging in GES-1 cells confirmed that TNF-α increased the mitochondrial localization of NOX2 (white arrows indicate colocalization). Nuclei (blue) were counterstained with DAPI. (F) The levels of and interaction between NOX2 and TOMM20 were determined by western blotting, and IP showed that Nec-1 decreased the epithelial expression of total and mitochondrial NOX2 in the primary epithelial cells of mice with PVL. TEM also revealed that Nec-1 partly normalized the abnormal or damaged mitochondria in the mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (G) The ATP concentration in the mouse models verified that Nec-1 and GSK2795039 (GSK, a NOX2 inhibitor) improved the production of ATP in mice with PVL. n = 6 per group. * P < 0.05. (H) Representative 4-HNE IHC staining (brown) and quantification analysis in SO mice and mice with PVL suggested that GSK2795039 (GSK) reduced the level of 4-HNE in mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (I) Representative MitoSox staining (red) and the MitoSox intensity in mouse model revealed that GSK2795039 alleviated mitochondrial ROS (mtROS) accumulation in the primary epithelial cells of mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (J) Related protein levels in primary epithelial cells from mouse models treated with the indicated agents ( shFADD , Nec-1 or BAY) showed interactions among TAK1, NF-κBp65, FADD and NOX2 signaling. (K) GES-1 cells were transfected with either the control vector or shFADD and then treated with TNF-α or vehicle, and the NOX2 luciferase reporter activities were presented (left panel). * P < 0.05 versus the vehicle group and TNF-α-treated cells transfected with shFADD . The NOX2 luciferase reporter activities in GES-1 cells transfected with pcDNA3.1-p65 -vector or pcDNA3.1 -vector were also shown (right panel), * P < 0.05.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Comparison, Microarray, Immunohistochemistry, Expressing, Imaging, Western Blot, Concentration Assay, Staining, Transfection, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: The alteration of glycolysis associated with dysfunction of the mitochondrial electron transport chain contributes to oxidative stress in the PHG. (A) Western blotting showed that the protein levels of LDHA and HK2 were increased in mice with PVL but were decreased by GSK2795039 (GSK, a NOX2 inhibitor) in mice with PVL. The ratios of the normalized LDHA/β-actin and HK2/β-actin densitometric units and the relative level of lactic acid were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL not treated with GSK2795039. (B) Oxygen consumption rate (OCR) analysis of primary epithelial cells isolated from mouse models suggested that increased nonmitochondrial oxygen consumption and reduced maximum respiration were present in mice with PVL but were reversed by GSK2795039. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (C) Examination of the glycolytic rate (for the extracellular acidification rate [ECAR]) showed that GSK2795039 treatment reduced basal and compensatory glycolysis in mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (D) Two-dimensional hierarchical clustering results of the genes of electron transport chain (ETC)-related elements between PHG patients (as PHG) and healthy volunteers (as Uninvolved) (n = 3 per group). (E) Western blotting analysis (left panel) of protein levels of representative ETC complex subunits in the primary epithelial cells of PHG patients and healthy volunteers (as Uninvolved) revealed that the levels of the mitochondrial complex I, II, III, IV and V subunits were decreased in PHG patients. The activity of the antioxidant enzyme SOD and the ratio of reduced (GSH) to oxidized (GSSG) states (GSH/GSSG) detected by assay kits (right panel) were found to be decreased in PHG. n = 6 per group. * P < 0.05. (F) Western blotting analysis of representative ETC complex subunits from primary epithelial cells isolated from mouse models suggested that Nec-1 (an inhibitor of RIPK1) mitigated the defects in ETC subunit expression and decreased ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (G) SOD activity and the GSH/GSSG ratio, as detected by assay kits, were inhibited in mice with PVL but were restored by Nec-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (H) Western blotting analysis and quantification of protein levels for representative ETC complex subunits from primary epithelial cells isolated from mouse models showed that Mdivi-1 reversed the decrease in mitochondrial complex I, II, III, IV and V subunit levels and reduced ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also presented. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment. (I) Decreases in SOD activity and the GSH/GSSG ratio detected by assay kits were found in mice with PVL but were reversed by Mdivi-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Western Blot, Isolation, Activity Assay, Expressing

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

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: