Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Tumor-intrinsic RRBP1 inhibition triggers antitumor immunity. ( A ) Representative images of IHC staining for RRBP1 and CD8 + T cells in BC samples. ( B ) The correlation between RRBP1 expression and CD8 + T-cell infiltration was analyzed based on 96 patients from in-house BC cohort. Scale bar: 50 µm. ( C ) Representative images of IHC staining for RRBP1 expression in PD, SD, PR, and CR samples. Scale bar: 50 µm. ( D ) Bar plot showed the response rates of anti-PD-L1 therapy. Blue bars represent CR/PR, Red bars represent PD/SD. ( E ) Volcano plot of RNA-seq data for shNC or shRRBP1 tumors (n=3). Differentially expressed genes were identified with the threshold of |log2 (fold change) | >1 and FDR<0.05. ( F ) GSEA for DEGs showed the activation of immune-associated pathways in shRRBP1 tumors in the RNA-seq data. ( G ) Representative images of IHC and mIHC staining for RRBP1 and CD8 + T cells in shNC, shRRBP1, control or radezolid tumor tissues. Expression levels of the indicated proteins were displayed. Scale bar: 20 µm. ( H, I ) Flow cytometry showed the percentages of CD8 + T cells in CD3 + cells in shNC, shRRBP1, control or radezolid tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using Spearman correlation analysis ( B ), unpaired two-tailed t-test ( I ). ****p<0.0001. BC, bladder cancer; CR, complete response; FDR, false discovery rate; progressive disease; PR, partial response; PD-L1, programmed death-ligand 1; RNA-seq, RNA sequencing; RRB1, ribosomal-binding protein 1; SD, stable disease; IHC, immunohistochemistry; GSEA, gene set enrichment analysis; DEGs, differentially expressed genes; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Immunohistochemistry, Expressing, RNA Sequencing, Activation Assay, Staining, Control, Flow Cytometry, Two Tailed Test, Binding Assay, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Single-cell RNA sequencing reveals the difference of CD8 + T-cell subgroup. The UMAP plot of CD8 + T cells subpopulation, color-coded by cell cluster and cell type. ( A ) The expression of markers in each CD8 + T cells subpopulation. ( B ) Bar plot showed the proportion of CD8 + T cells subpopulation in the shNC and shRRBP1 groups. ( C ) The percentage of each CD8 + T-cell clusters in shNC and shRRBP1 groups. ( D ) Heatmap showed the differentially activated pathway among all the CD8 + T-cell clusters. ( E ) The differentially expressed genes in CD8 + T cells between shNC and shRRBP1 groups. ( F ) KEGG analysis for differentially expressed genes showed the enrichment of immune-associated pathways. ( G, H ) mIHC and flow cytometric analysis displayed the tumor-infiltrating IFN-γ + or GZMB + CD8 + T cells in shNC or shRRBP1 tumor tissues. Scale bar: 20 µm. ( I–K ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Isotype control (IgG) or anti-mouse CD8 antibody administered on days –6, –3, and –1 before tumor challenge, with the same dose repeated on days 7, 9 and 11 after tumor challenge. Tumor sizes ( I ), volumes ( J ), and weight ( K ) were measured. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( H, K ) and two-way ANOVA with Tukey’s multiple comparison test ( J ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; GZMB, Granzyme B; IFN, interferon; TEX, exhausted T cells; UMAP, Uniform Manifold Approximation and Projection; mIHC, multiplex immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Single Cell, RNA Sequencing, Expressing, Injection, Control, Two Tailed Test, Comparison, Multiplex Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition promotes antitumor immunity via the CXCL10-CXCR3 axis in BC. ( A ) ScRNA-seq data showed the CXCR3 expression of CD8+T cells in shNC and shRRBP1 groups. ( B ) The correlation between CXCR3 expression and CXCL10 expression or activated CD8 + T cell based on 571 patients from TCGA-BLCA cohort and GSE13507 cohorts. ( C ) MB49 cells were co-cultured with CD8 + T cells, and tumor cells were stained with crystal violet. ( D ) Evaluation of the effect of genetic inhibition of RRBP1 on the cytotoxicity of CD8 + T cells in vitro conditioned culture model. ( E ) Schematic diagram of in vitro CD8 + T-cell migration assays. ( F ) The number of CD8 + T cells passing through the membrane of a Transwell system was analyzed by flow cytometry. ( G–I ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice received intraperitoneal injection of either vehicle or anti-CXCL10 when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( G ), volumes ( H ), and weights ( I ) were measured. ( J ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( K ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( D, F, I, K ) and two-way ANOVA with Tukey’s multiple comparison test ( H ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; RRBP1, ribosomal-binding protein 1; scRNA-seq, single-cell RNA sequencing; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry; BLCA, bladder urothelial carcinoma.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Cell Culture, Staining, In Vitro, Migration, Membrane, Flow Cytometry, Injection, Two Tailed Test, Comparison, Binding Assay, Single Cell, RNA Sequencing, Immunohistochemistry, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition enhances response to anti-PD-L1 therapy in BC. ( A–D ) The protein expression of surface PD-L1 was analyzed in BC cells or tumor tissues by flow cytometry after RRBP1 inhibition and was shown as the mean fluorescence intensity. ( E–G ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice were received intraperitoneal injection of either vehicle or anti-PD-L1 antibody when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( E ), volumes ( F ), and weights ( G ) were measured. ( H ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( I ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( B, D, G, I ) and two-way ANOVA with Tukey’s multiple comparison test ( F ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; PD-L1, programmed death-ligand 1; RRBP1, ribosomal-binding protein 1; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Injection, Staining, Two Tailed Test, Comparison, Binding Assay, Immunohistochemistry, Multiplex Assay

Journal: Cells

Article Title: Identification of a Potent Cytotoxic Pyrazole with Anti-Breast Cancer Activity That Alters Multiple Pathways

doi: 10.3390/cells11020254

Figure Lengend Snippet: P3C cell death induction is mediated through apoptosis via intrinsic and extrinsic pathways. ( A ) Evaluation of phosphatidylserine externalization through flow cytometry indicated that P3C induced apoptosis in MDA-MB-231 cells. Analysis was performed after 24 h of exposure to P3C. Percentages shown represent the total apoptosis values (early and late apoptosis, ); ( B , C ) P3C-treated MDA-MB-231 cells exhibited a significant amount of caspase-8 activation after 4 h of incubation , whereas caspase-3/7 activation was detected after 8 h of incubation . Cells with a positive fluorescence for NucView 488 caspase-3 enzyme substrate denoted caspase-3 activation, whereas, cells indicating positive fluorescent levels of FITC-IETD-FMK marker designated caspase-8 activation, both measurements analyzed through flow cytometry. For these experiments ( A – C ), cells were treated with P3C CC 50 (5.52 µM) and 2× CC 50 (11.04 µM), also 1% DMSO, 1 mM H 2 O 2 as vehicle and positive control for cytotoxicity, respectively, as well as untreated controls. Averages of three technical replicates are shown in percentages, and standard deviations are denoted on each bar. Statistical evaluation on data was accomplished by using two-tailed Student’s paired t -test, and the asterisk annotations in each graph represent statistical significance of the treatments against the vehicle control (**) p < 0.01 and (***) p < 0.001.

Article Snippet: To monitor the activation of caspase-3/7 in live cells, the NucView 488 caspase-3/7 enzyme-substrate (Biotium, Hayward, CA, USA) and flow cytometry methods were employed.

Techniques: Flow Cytometry, Activation Assay, Incubation, Fluorescence, Marker, Positive Control, Two Tailed Test

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) CD5L-KO mice were generated by CRISPR-Cas9 engineering. Schematic representation of the genome editing strategy to silence Cd5l expression by inserting three in-frame stop codons and a frame shift. (B) Quantification by ELISA of CD5L in the sera of C57BL/6 WT and CD5L-KO mice. DL: detection limit (6.25 pg/mL). Data from at least 3 independent experiments (Mann-Whitney test). (C) Intracellular CD5L was detected, after permeabilization of the cells with Triton X100, by staining with goat anti-CD5L polyclonal antibody followed by Alexa 488-coupled donkey anti-goat secondary antibody. Peritoneal macrophages from WT or CD5L-KO healthy mice were identified through staining with anti-mouse F4/80 mAb followed by Alexa 594-coupled anti-rat secondary antibody. DAPI was used as a nuclear counterstaining. Scale bar: 10 μm. (D) WT or CD5L-KO mice were subjected to surgery to expose and ligate the cecum at approximately half the distance between the distal pole and the base of the cecum, and through-and-through puncture with a 21G needle, to induce a mid-grade sepsis condition. N = 15 (each group). Kaplan–Meier survival curves were generated to compare mortality between the two groups and significance was determined by log-rank (Mantel-Cox) test. (E) Body weight loss quantification. ***, p < 0.005.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Generated, CRISPR, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: WT or CD5L-KO mice were subjected to CLP to induce mid-grade sepsis. For that, animals were subjected to surgery, exposure and ligation of the cecum (corresponding to approximately half the distance between the distal pole and the base of the cecum) and through-and-through puncture with a 21G needle. Control mice (sham) were subjected to the same surgical procedure but without cecum ligation and puncture. Mice were euthanized 6 or 24 h post-surgery. (A) CFU counts from the peritoneal cavity, blood, lung, liver and kidney. (B) Absolute counts assessed by flow cytometry of cellular populations in the peritoneal cavity. Subsets analyzed: leukocytes, CD45 + ; neutrophils, CD45 + CD11b + Ly6G + ; macrophages, CD45 + CD11b + CD11c - F4/80 + . Ly6C and CD206 markers were used to distinguish between M1 and M2 within the macrophage population. (C) Ly6G and CD11b mean fluorescence intensity (MFI) in expressing neutrophils obtained by geometric mean statistics. (D) Lung, liver and kidney tissue sections obtained 24 h post CLP were stained with hematoxylin and eosin. Scale: 100 μm (E) Pathology score of lung, liver and kidney 24 h post CLP in a double blinded analysis. 0, no signs of inflammation; 1, minimal inflammatory signs; 2, mild inflammation; 3, moderate to severe inflammation. (F) The indicated cytokines were quantified by a multiplex bead-based immunoassay in samples from the peritoneal cavity (left panel) and blood serum (right panel). Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05; **, p < 0.01; ns, not statistically significant.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Ligation, Flow Cytometry, Fluorescence, Expressing, Staining, Multiplex Assay, Bead-based Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) Quantification, by ELISA, of CD5L in the peritoneal cavity (left panel) and serum (right panel) of C57BL/6 mice at 0, 6 or 24 h after mid-grade sepsis (CLP) or in sham operated (sham) animals. (B) WT and CD5L-KO naïve healthy mice were subjected to mid-grade CLP and 3 h later were IV administered with recombinant CD5L (rCD5L) at 0 (PBS) or 2.5 mg/Kg (CD5L). Mice were euthanized 1 h after injection to recover the fluids. Amount of CD5L in the peritoneal cavity (left panel) and serum (right panel), quantified by ELISA. (C) Scatter graphs with detailed illustration of the relation between peritoneal and blood CD5L with the indication of Spearman’s rank correlation in both WT and CD5L-KO mice upon CD5L IV injection as detailed above. Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05; **, p < 0.01; ns, not statistically significant.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Injection, IV Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) Percentage of thioglycolate-elicited Ly6G + CD11b + neutrophils among all cells collected from the peritoneal cavity of WT and CD5L-KO mice, determined by flow cytometry. (B) pHrodo™ Red E. coli BioParticles™ were incubated with 0 or 10 μg of rCD5L for 1 h on ice in TBS containing Ca 2+ . After washing, samples were resuspended in Laemmli buffer and heated at 95 °C for 10 min. Western blot detection of CD5L bound to the particles, using mouse anti-His mAb followed by a secondary goat anti-mouse HRP antibody. (C-D) Phagocytosis of pHrodo particles. Neutrophils were cultured in the presence of pHrodo particles pre-coated with 0 or 10 μg of rCD5L. (C) Percentage of Ly6G + CD11b + neutrophils that internalized pHrodo particles, quantified by flow cytometry. (D) MFI values (geometric mean) of pHrodo channel within the pHrodo + neutrophil population. (E) Cecal bacteria were incubated with 0, 2 and 10 μg of rCD5L for 1 h on ice in TBS containing Ca 2+ . Detection of bacteria-bound rCD5L as in (B). (F) Neutrophils from WT or CD5L-KO mice were cultured in the presence of cecal bacteria pre-coated with 0, 2 or 10 μg of rCD5L. After 3 h, cells were washed, lysed and plated for CFU enumeration. (G) Intracellular staining of CD5L in WT or CD5L-KO naïve mouse blood neutrophils (Ly6G + CD11b + ) and monocytes (F4/80 + CD11b + ) was performed after fixation and permeabilization, with goat anti-mCD5L polyclonal antibody followed by Alexa 488-coupled donkey anti-goat secondary antibody and analyzed by flow cytometry. Data from at least 2 independent experiments. Mann-Whitney test did not reveal any statistical difference between relevant groups.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Incubation, Western Blot, Cell Culture, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) CD5L-KO mice were IV injected with 2.5 mg/Kg of rCD5L, and the circulating amount of rCD5L was quantified by ELISA at the indicated time-points. (B) Protocol for analysis of the immune response after CLP and rCD5L IV treatment. Top, analysis at 6 h, mice had been injected IV with 2.5 mg/Kg rCD5L at 3 h post-surgery, or PBS (untreated), and euthanized 3 h later. Bottom, analysis at 24 h, mice were IV injected with 2 doses of 2.5 mg/Kg rCD5L at 3 and 6 h after surgery, or PBS (untreated), followed by euthanasia at 24 h. (C) Absolute counts and frequencies of cellular populations in the peritoneal cavity, measured by flow cytometry. Subsets analyzed: leukocytes, CD45 + ; neutrophils, CD45 + CD11b + Ly6G + ; macrophages, CD45 + CD11b + CD11c - F4/80 + . Ly6C and CD206 markers were used to distinguish between M1 and M2 within the macrophage population. (D) Ly6G and CD11b MFI in expressing neutrophils, obtained by geometric mean statistics. (E) CFU counts of bacteria obtained from the peritoneal cavity and blood, grown in aerobic or anaerobic conditions, or from lung, liver and kidney, grown in aerobiosis. (F) The indicated cytokines were quantified by a multiplex bead-based immunoassay in samples from the peritoneal cavity (upper panels) and blood serum (lower panels). Data from at least 2 independent experiments (Mann-Whitney test). (G) C57BL/6 mice were subjected to CLP to induce highgrade sepsis. At 3 and 6 h after the surgery, mice were injected IV with doses of 2.5 mg/Kg of rCD5L. Kaplan–Meier survival curves were generated to compare mortality between the two groups and significance was determined by log-rank (Mantel-Cox) test. The number of mice per group is indicated (n). *, p < 0.05; ****, p < 0.0001.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Multiplex Assay, Bead-based Assay, MANN-WHITNEY, Generated

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) WT and CD5L-KO mice were subjected to CLP to induce mid-grade sepsis, and CXCL1 was quantified in the peritoneal cavity or blood serum 3 h or 6 h after surgery. (B) WT C57BL/6 mice were subjected to CLP to induce high grade sepsis and IV injected with 2.5 mg/Kg of rCD5L, or with PBS (untreated) 3 h after surgery. CXCL1 was quantified by a multiplex bead-based immunoassay in the peritoneal cavity and blood serum 6 h after CLP. Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Injection, Multiplex Assay, Bead-based Assay, MANN-WHITNEY

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 1 Proliferation and specific cytotoxic effects of CART-19 cells. A The design of the CAR-T cell construction experiments. B Morphological images of activated T cells clustered after 24 h and 72 h of incubation with TransAct CD3/28 beads. C Flow cytometric analysis of CAR expression on the surface of mock T, and CART-19 cells with biotin-conjugated anti-Fab antibody followed by PE-conjugated streptavidin. Gating was based on the same cells stained with isotype-matched antibody. The median fluorescence intensity (MFI) was calculated for CAR-T population in the PE fluorescence channel (right column). This result is the representative of three separate experiments using cells from healthy volunteer donors. D The phenotypic characterization of CART-19 cells by flow cytometry. The ratio of CD4+ / CD8+ T cells (left) and the proportion of TN/CM (right) are shown. E Growth curves of CAR-T cells. Data represent the mean ± s.d. of three separate experiments. F Cytolytic activities of CART-19 cells in cell assays. Nalm-6 cells were labeled with CFSE labeling reagent (Sigma-Aldrich, USA) and co-cultured with CART-19 cells at the E: T ratio of 1:1 for 30 h. The presence of CFSE-labeled cells was observed by mi croscopy. Bar, 100 μm. G Cytotoxic activity of mock NT and CART cells against Nalm-6 cells. The effector cells were co-cultured with target cells at E: T ratios of 1:5, 1:2, 1:1 and 5:1 with a total cell number of 1 × 106. H Dynamic changes of cytokine secretion profile of CART-19 cells during 24 h after co-culture with Nalm-6 cells at E: T ratios of 1:5 to 5:1. Data were visualized by heatmap. Concentrations (pg/ml) of cytokines and chemokines in the supernatant were detected by multiplex immunoassay and the values were log2 transformed

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Incubation, Expressing, Staining, Fluorescence, Flow Cytometry, Labeling, Cell Culture, Activity Assay, Co-Culture Assay, Multiplex Assay, Transformation Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: To evaluate CAR expression after 7–10 days of culture, CART-19 cells were washed once and incubated with goat anti-human biotin conjugated anti-Fab antibody (Jackson ImmunoResearch, USA) for 30 min at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) A schematic depicting our strategy to generate GBA1 variants with enhanced secretion. Endogenous signal sequence of human GBA1 was swapped with signal sequences from highly secreted proteins. Top 4 signal sequences (SS) were narrowed down using in-silico tools that predicted robust secretion as well as high (>96%) probability of cleavage at the end of signal sequence. (B) HEK293T cells were transfected with GBA1 plasmid containing the indicated SS variants, lysed, and GCase enzyme activity was determined in cell lysates. Mean ± SEM, n = 4 independent experiments. Untransfected versus all GBA1 constructs as well as GFP transfected versus all GBA1 constructs: ***p<0.001; One-way ANOVA with Turkey’s multiple comparison test. (C) Representative immunofluorescence images demonstrating co-localization of active GCase and lysosomes. HEK293T cells were transfected with GBA1 variant constructs, incubated with MDW933 fluorescence probe to label active GCase, and immunostained with Lamp1 antibody for lysosomes. Blue and yellow arrowheads point to individual puncta showing co-localization of GCase with Lamp1. Scale bar is 10 microns. (D and E) HEK293T cells were transfected with GBA1 variant constructs and cell culture media was collected to detect secreted GCase (D) and measure GCase enzyme activity (E). Mean ± SEM, n = 4 independent experiments. Untransfected versus all GBA1 constructs as well as GFP transfected versus all GBA1 constructs: ***p<0.001; One way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Sequencing, In Silico, Transfection, Plasmid Preparation, Activity Assay, Construct, Comparison, Immunofluorescence, Variant Assay, Incubation, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: AAV GMU01 capsid expressing the indicated GBA1 variants were administered by bilateral ICV to 4 month-old C57/BL6 mice at 1e11 vector genomes per animal and 5 μl per hemisphere. N=4 animals per group. The sagittal sections of brain hemisphere were analyzed 4 weeks post-injection. (B) Representative images demonstrating GCase secretion with engineered variants. Vector biodistribution is shown with in situ hybridization to WPRE (top panels) and GCase is shown with immunohistochemistry. (C) Representative images demonstrating cross-correction. Higher magnification images of WPRE mRNA (left) and GCase protein (right) in SS3-GBA1 injected mice brain, image corresponds to the purple box in (A). Cells positive for both WPRE mRNA and GCase are shown in red arrows (AAV-transduced cells) while mRNA negative and GCase positive cells are shown in green arrows (cross-corrected cells). Scale bar is 500 microns.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Expressing, Plasmid Preparation, Injection, In Situ Hybridization, Immunohistochemistry

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Measurement of Lyso-GL1 lipid substrate levels by LC-MS in cortex and liver of C57/BL6 mice 24 hours post CBE injection by IP injection at indicated doses. N=7 animals for CBE 0 mg/kg and N=4 per all the other groups. Data are Mean ± SEM. (B) Kinetic analysis of Lyso-GL1 lipid substrate by LC-MS in cortex and liver of 8-month-old mice from three different genotypes ( Gba D409V/D409V , Gba D409V/+, and Gba +/+ ). Mice with 100 mg/kg CBE IP dosing. Data are Mean ± SEM. 8 – 10 animals at each time point. (C) Study design: AAV GMU01 capsid expressing the indicated GBA1 variants were administered by bilateral ICV to 4-month-old C57/BL6 mice at 1e11 vector genomes per animal and 5μl per hemisphere. N=8 animals per group. CBE (conduritol β-epoxide) was administered via IP injection at 100 mg/kg 24 hours prior to necropsy. (D) Representative image of in situ hybridization to WPRE mRNA from the analyzed sagittal sections. Cortex is proximal to site of injection and cerebellum is distal to site of injection. (E-G) Measurement of Lyso-GL1 lipid substrate levels by LC-MS in midbrain (E), cerebellum (F), and hindbrain (G). No CBE group in the graphs shown is control mice that did not receive any AAV vector or CBE injection. 8 mice per group. ***p<0.001, **p<0.01, *p<0.05; One way- ANOVA with Tukey’s multiple comparisons test with all groups compared to CBE-treated vehicle injected group.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Liquid Chromatography with Mass Spectroscopy, Injection, Expressing, Plasmid Preparation, In Situ Hybridization, Control

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 2-3 years old cynomolgus monkeys (2-3kg) were dosed either with AAV GMU01-WT-GBA1 or AAV GMU01-SS3-GBA1 at 1.25e13 vector genomes per animal by direct injection to the cisterna magna (ICM) in 2.5 ml volume at 0.125 ml/min rate. 6 weeks post- dosing, 30 mg/kg of CBE was administered by IV injection 48 hours prior to necropsy. Samples represent 64 brain biopsy punches encompassing 19 distinct grey matter regions and 7 distinct white matter regions. (B and C) AAV vector genome copies determined from 64 brain biopsy punches by Gba1 dPCR and normalized to the Tubb3 gene copy number to obtain VG copies per cell (B). Transgene (mRNA) expression measured by GBA1 RT-dPCR normalized to endogenous Hprt gene (C). Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions. (D) Correlation of vector genome with transgene expression (mRNA) was determined between WT-GBA1 and SS3-GBA1. Each data point is average of all NHPs for that punch. Non- parametric Spearman’s rank correlation. (E) Lyso-GL1 changes in plasma pre-AAV, post-AAV and pre-CBE, and at necropsy across all NHPs. (F and G) Lyso-GL1 level (F) and C18 GL1 level (G) across 64 brain biopsy punches. Each data point is average of all NHPs in the group for that punch. ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (H and I) Multiplex fluorescent imaging assay with in situ hybridization for mRNA and immunohistochemistry for GBA1 protein and cell marker. Low magnification image showing stained motor cortex region of SS3-GBA1 injected NHP and zoomed-in images corresponding to the white box (H). Triplex to determine identity of cross-corrected cells using specific cell type markers, NeuN for neurons, S100b for astrocytes, and Iba1 for microglia (I). Yellow arrowheads point to AAV-transduced cells and red arrowheads point to cross-corrected cells. Scale bar is 20 microns.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Plasmid Preparation, Injection, IV Injection, Expressing, Clinical Proteomics, Multiplex Assay, Imaging, In Situ Hybridization, Immunohistochemistry, Marker, Staining

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 2-3 years old cynomolgus monkeys (2-3kg) were administered with 3 different doses of AAV GMU01-SS3-GBA1 by direct injection to the cisterna magna (iCM) in 2.5 ml volume at 0.125 ml/min rate, 2.5e12 vector genomes per animal (low dose), 7.5e12 vector genomes per animal (mid dose), and 2.5e13 vector genomes per animal (high dose). 8 weeks post-dosing, 30 mg/kg of CBE was administered by IV injection 24 hours prior to necropsy. Samples represent 64 brain biopsy punches encompassing 19 distinct grey matter regions and 7 distinct white matter regions. (B and C) Assessment of vector genomes in dose-range finding study with 3 doses tested, 5 NHPs per group. Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions (B). Same data shown across different brain regions (C). T/I/C: temporal/insulate/cingulate (D) GCase enzyme activity in dose-range finding study with 3 doses tested, N=5 NHPs per group. Median with inter-quartile range across 64 punches representing 19 grey matter and 7 white matter regions. **p<0.05, ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (E and F) Lipidomics performed in 3 NHPs per group. Lyso-GL1 level measured in plasma (E) and from 47 brain biopsy punches from 19 grey matter regions. Median with inter-quartile range across punches. ****p<0.0001, ***p<0.001; Two way-ANOVA with Tukey’s multiple comparisons test. (G – J) Histopathological analyses in NHPs with AAV.GMU01 SS3-GBA1 dose-range finding study. Histopathological findings reported with severity scores across both central nervous system and peripheral tissues by board certified clinician. Scores reported for brain (G), spinal cord (H), DRGs (I), and sciatic nerve (J). Data are Mean ± SEM. Each dot is score for individual NHP. (K) Quantification of human GBA1 protein levels in 12 brain regions from N=10 healthy human donor brain tissues (aged 55-to-75 years old) by LC-MS. Human tissue was obtained from the NIH Neurobiobank at the University of Maryland, Baltimore, MD and the Sepulveda Research Corporation. Data are Mean ± SEM. Each data point represents brain biopsy punches from each human donor. CC: corpus callosum; SN: substantia nigra; DN: dentate nucleus; Th: Thalamus, lateral nuclear group; Cb: cerebellum; PWM: periventricular white matter; Hp: hippocampus; BA: Brodmann area. (L) Quantification of human GBA1 protein levels from 3 NHPs per group. Median with inter- quartile range across 32 punches from NHPs plotted against the 12 punches from human donors. ***p<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Injection, Plasmid Preparation, IV Injection, Activity Assay, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: AAV GMU01-SS3-GBA1 was administered by bilateral ICV to 4-month-old Gba D409V/+ mice at 1.6e11 vector genomes per animal and 4μl per hemisphere. 6-12 animals per group. Brain, plasma, and CSF were analyzed 3-month, 6-month, and 9-month post-injection. CBE (conduritol β-epoxide) was administered at 100 mg/kg 24 hours prior to each timed necropsy. (B) Representative image of in situ hybridization to WPRE mRNA from the analyzed sagittal sections. (C – F) Vector genome assessment of the longitudinal pharmacology study with in-life duration of 3-month, 6-month, and 9-month post AAV-dosing. AAV vector genome copies (left) and Lyso- GL1 lipid clearance (right) in cortex (C), sub-cortex (D), cerebellum (E), and hindbrain (F) across all mice in the study. 6 animals for No CBE group, 12 animals for Vehicle group, and 12 animals for SS3-GBA1 group. Data are Mean ± SEM. ***p<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test. (G) Lyso-GL1 lipid clearance in plasma of SS3-GBA1 injected mice. Data is Mean ± SEM. ***P<0.0001; Two way-ANOVA with Tukey’s multiple comparisons test.

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Plasmid Preparation, Clinical Proteomics, Injection, In Situ Hybridization

Journal: bioRxiv

Article Title: AAV gene therapy for GBA-PD and Gaucher Disease

doi: 10.1101/2025.06.17.660133

Figure Lengend Snippet: (A) Study design: 3-month-old C57/BL6 mice were injected with 4e13 VG/kg of vehicle or AAV GMU01-SS3-GBA1 intravenously. AAVs were expressed for 4 weeks followed by 100mg/kg of CBE IP injection 24 hours prior to necropsy. (B) Vector genome assessed in different visceral organs such as liver, spleen, heart, and soleus muscle. 8 animals per group. Data is Mean ± SEM. (C) Schematic view of liver sectioning strategy and representative images demonstrating robust GBA1 secretion. Vector biodistribution is shown with in situ hybridization to WPRE (top panels) and GBA1 protein expression is shown with huGBA1 immunohistochemistry after 4 weeks of expression. (D – G) Quantification of human GBA1 protein level by ELISA and Lyso-GL1 lipid clearance by LC-MS in liver (D), spleen (E), heart (F), and soleus muscle (G). 8 animals per group, No CBE group in the graphs shown is control mice that did not receive any AAV vector or CBE injection. Data are Mean ± SEM. *p<0.05, ***p<0.0001; unpaired Student’s t-test (human GBA1 protein) and ***p<0.0001; One way-ANOVA with Dunnett’s multiple comparisons test (Lyso-GL1). All groups compared to vehicle group. (H) Evaluation of vector exposure, GCase enzyme activity, and Lyso-GL1 lipid clearance from bone marrow. 8 animals per group and No CBE group in the graphs shown is control mice that did not receive any vector or CBE injection. Data are Mean ± SEM. ***p<0.0001; unpaired Student’s t-test (VGs/cell) and ***p<0.001; One way-ANOVA with Tukey’s multiple comparisons test (GCase activity and Lyso-GL1).

Article Snippet: The slides were then incubated with human GBA1 antibodies (1:100, abcam, ab125065 and 1:400, Novus, NBP2-45829) for rodent and NHP, respectively.

Techniques: Injection, Plasmid Preparation, In Situ Hybridization, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy, Control, Activity Assay

Journal: Experimental & Molecular Medicine

Article Title: Cigarette smoke extract enhances neutrophil elastase-induced IL-8 production via proteinase-activated receptor-2 upregulation in human bronchial epithelial cells

doi: 10.1038/s12276-018-0114-1

Figure Lengend Snippet: a BEAS-2B cells were treated with vehicle control (VC) or NE (1 U/ml) for the indicated times. Total cellular extracts were subjected to western blot analysis for p-ERK and GAPDH. b Cells were pre-treated with an MEK inhibitor (U0126, 20 μM) for 1 h and then stimulated with VC or NE (1 U/ml) for 24 h. Levels of IL-8 in cell supernatants were measured by multiplex bead assay. Data represent the mean ± SD; ** P < 0.05. c – e BEAS-2B cells were transiently transfected with control siRNAs or PAR2 siRNAs using a Neon electroporation kit. Forty-eight hours after transfection, cells were stimulated with VC or NE for 1, 2, 4 h ( d ) or 24 h ( e ). The expression of PAR2 was measured by quantitative real-time PCR. Data were normalized to the expression of GAPDH. Data represent the mean ± SD; ** P < 0.05. Total cellular extracts were subjected to western blot analysis for PAR2, p-ERK and GAPDH. IL-8 concentrations in culture media were measured by multiplex bead assay. Data represent the mean ± SD; ** P < 0.05

Article Snippet: The primer information is as follows: PAR2 (Hs00608346_m1) and GAPDH (Hs99999905_m1).

Techniques: Control, Western Blot, Multiplex Assay, Transfection, Electroporation, Expressing, Real-time Polymerase Chain Reaction

Journal: Experimental & Molecular Medicine

Article Title: Cigarette smoke extract enhances neutrophil elastase-induced IL-8 production via proteinase-activated receptor-2 upregulation in human bronchial epithelial cells

doi: 10.1038/s12276-018-0114-1

Figure Lengend Snippet: a BEAS-2B cells were treated with CSE (0.5 and 1%) for the indicated times. The expression of PAR2 was measured by quantitative real-time PCR. Data were normalized to the expression of GAPDH. Data represent the mean ± SD; ** P < 0.05. b Cells were stimulated with CSE (1%) for 24 h. The cells were fixed and permeabilized for 10 min. Immunofluorescent staining of PAR2 was performed using an anti-PAR2 antibody, followed by an Alexa Fluor 488 antibody. Cells were analyzed using an ECLIPSE TE300 (Nikon) fluorescence microscope. The mean intensity of PAR2 per cell number was determined using ImageJ. c , d BEAS-2B cells were treated with CSE (0.5, 1, and 2%) or NE (1 U/ml) for the indicated times. Total cell lysates were extracted and subjected to western blot analysis for PAR2 and GAPDH. e , f Cells were treated with CSE (0.5, 1, 2, and 4%) or NE (1 U/ml) for 24 h. The membrane fraction was isolated and then subjected to western blot analysis for PAR2 using an antibody to detect the N-terminal extracellular domain of PAR2. Results are representative of three independent experiments

Article Snippet: The primer information is as follows: PAR2 (Hs00608346_m1) and GAPDH (Hs99999905_m1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence, Microscopy, Western Blot, Membrane, Isolation

Journal: Experimental & Molecular Medicine

Article Title: Cigarette smoke extract enhances neutrophil elastase-induced IL-8 production via proteinase-activated receptor-2 upregulation in human bronchial epithelial cells

doi: 10.1038/s12276-018-0114-1

Figure Lengend Snippet: a BEAS-2B cells were treated with CSE (1%) for the indicated times. Total cellular extracts were subjected to western blot analysis for p-ERK, p-p38, p-Akt, and GAPDH. b Cells were pre-treated with an MEK inhibitor (U0126, 2 or 10 μM), PI3K/Akt inhibitor (LY294002, 10 μM), or p38 inhibitor (SB203580, 10 μM) for 2 h and then stimulated with CSE for 24 h in the presence of U0126, LY294002, or SB203580. Membrane and cytoplasmic proteins were extracted and subjected to western blot analysis for PAR2 and heat shock protein 90 (Hsp90). c BEAS-2B cells were pre-treated with SB203580 (10 μM) for 2 h and then stimulated with CSE (1%) in the presence or absence of SB203580 for 24 h. The cells were stimulated with VC or NE (1 U/ml) for 24 h in the absence of SB203580 and CSE. IL-8 concentrations in cell supernatants were measured by multiplex bead assay. Data represent the mean ± SD; ** P < 0.05

Article Snippet: The primer information is as follows: PAR2 (Hs00608346_m1) and GAPDH (Hs99999905_m1).

Techniques: Western Blot, Membrane, Multiplex Assay

Journal: Experimental & Molecular Medicine

Article Title: Cigarette smoke extract enhances neutrophil elastase-induced IL-8 production via proteinase-activated receptor-2 upregulation in human bronchial epithelial cells

doi: 10.1038/s12276-018-0114-1

Figure Lengend Snippet: a Experimental protocols for a murine model of emphysema. C57BL/6 mice were intratracheally instilled with vehicle or CSE as described in the materials and methods section. Mice ( n = 4 per group) were sacrificed at week 8 after first instillation to isolate lungs. b Lung homogenates from mice were subjected to western blot analysis for PAR2 and GAPDH. Gel data were quantified using Scion image densitometry (right panel). Data represent the mean ± SE; ** P < 0.05. c PAR2 immunohistochemistry in the lung tissues from mice. PAR2 was increased in lung epithelial cells of CSE-treated mice (arrows). d Total lung lysates obtained from normal ( n = 4), smokers ( n = 4), and COPD patients ( n = 4) ( c ) were subjected to western blot analysis for PAR2 and GAPDH. Gel data were quantified (right panel). Data represent the mean ± SE; ** P < 0.05. e PAR2 immunohistochemistry in the lung tissues from human patients. Arrows identify the epithelial cells. Original magnifications, ×200

Article Snippet: The primer information is as follows: PAR2 (Hs00608346_m1) and GAPDH (Hs99999905_m1).

Techniques: Western Blot, Immunohistochemistry

Journal: Developmental cell

Article Title: Xist Repeats A and B account for two distinct phases of X-inactivation establishment

doi: 10.1016/j.devcel.2020.05.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Labeled oligo probe pool (1–5 nM for Xist RNA FISH, 100 nM for Atrx or Mecp2 nascent RNA FISH) was added to hybridization buffer containing 25% formamide, 2x SSC, 10% dextran sulfate, and nonspecific competitor (0.1 mg/mL mouse Cot-1 DNA [Thermo Fisher Scientific]).

Techniques: Recombinant, Isolation, Multiplex Assay, Software

Journal: Scientific Reports

Article Title: Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens

doi: 10.1038/s41598-024-58372-5

Figure Lengend Snippet: Murine multi-tissue block & IHC images. ( A ) Shows an image of an H&E stained section from a multi-tissue block, containing CT26, 4T1 and B16F10 tumors and a transverse section of normal mouse spleen. Scale bar equals 2 mm. ( B ) Shows representative IHC images of CD3ε, CD4, CD8α and FoxP3 in normal mouse spleen (top row) and in CT26 tumor (bottom row). Scale bar equals 25 microns. ( C ) Shows merged (upper left) and individual immunofluorescence channels of CT26 tumor that was immunolabeled with the murine multiplex panel. Scale bar equals 25 microns.

Article Snippet: CT26, 4T1 and B16F10 cell lines were purchased from ATCC (Manassas, VA).

Techniques: Blocking Assay, Staining, Immunofluorescence, Immunolabeling, Multiplex Assay

Journal: Scientific Reports

Article Title: Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens

doi: 10.1038/s41598-024-58372-5

Figure Lengend Snippet: Murine 4-plex panel: concordance assay. ( A ) Shows the design of the concordance assay for the murine 4-plex panel. Five serial sections are cut from the multi-tissue block. The third section is immunolabeled with the 4-plex assay while the remaining sections are 1-plex assays immunolabeled for CD3ε, CD4, FoxP3 and CD8α. ( B ), ( C ) and ( D ) Show % positive cells quantified through whole-slide image analysis from the 1-plex and 4-plex images of CT26, 4T1 and B16F10 tumors, respectively. ( E ) Shows a Bland–Altman plot of the relative difference in % positive cells between the 1-plex image and the 4-plex image for all the murine tumor models. Here each data point pertains to one biomarker and the color pertains to the tumor model type. ( F ), ( G ), ( H ) and ( I ) Show the average intensity/cell of FoxP3, CD4, CD8α and CD3ε biomarkers, respectively, in the 1-plex and 4-plex images for different murine tumor models. ( J ) Shows a Bland–Altman plot of the relative difference in the average intensity/cell for the different biomarkers across all three murine tumor models. Here each data point pertains to one biomarker and the color pertains to the tumor model type.

Article Snippet: CT26, 4T1 and B16F10 cell lines were purchased from ATCC (Manassas, VA).

Techniques: Blocking Assay, Immunolabeling, Plex Assay, Biomarker Discovery

Journal: Scientific Reports

Article Title: Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens

doi: 10.1038/s41598-024-58372-5

Figure Lengend Snippet: Murine 4-plex panel: reproducibility assay. ( A ) Shows the design of the reproducibility assay. Fifteen serial sections were cut from the multi-tissue block and immunolabeled with the murine 4-plex panel in 5 separate runs with 3 replicates per run. ( B ) Shows representative fluorescence images of the biomarkers across the different runs. Scale bar equals 50 microns. ( C ), ( D ) and ( E ) Show % positive cells of single-marker cell phenotypes for CT26, 4T1 and B16F10 tumor models, respectively, while ( F ), ( G ) and ( H ) show the same for multiple-marker cell phenotypes (T helper : CD3ε+CD4+ cells; T reg : CD3ε+CD4+FoxP3+ cells; T cyt denotes CD3ε+CD8α+ cells). ( I ), ( J ) and ( K ) Show the intra-run and inter-run CV for single-marker and multi-marker cell phenotypes for CT26, 4T1 and B16F10 tumor models, respectively.

Article Snippet: CT26, 4T1 and B16F10 cell lines were purchased from ATCC (Manassas, VA).

Techniques: Blocking Assay, Immunolabeling, Fluorescence, Marker

Journal: Scientific Reports

Article Title: Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens

doi: 10.1038/s41598-024-58372-5

Figure Lengend Snippet: Local thresholding decreases inter-run variability without affecting intra-run variability. ( A ) and ( B ) Show the % positive cells for single-marker and multi-marker cell phenotypes, respectively, for the CT26 murine tumor model across different runs. ( C ) Shows the intra-run and inter-run CVs of single- and multi-marker cell phenotypes for the CT26 tumor model. ( D ), ( E ) and ( F ) Show the % positive cells for single-marker ( D ) and multi-marker ( E ) cell phenotypes, and intra-run and inter-run CVs of single- and multi-marker cell phenotypes ( F ) for the 4T1 tumor model.

Article Snippet: CT26, 4T1 and B16F10 cell lines were purchased from ATCC (Manassas, VA).

Techniques: Marker

Journal: Scientific Reports

Article Title: Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens

doi: 10.1038/s41598-024-58372-5

Figure Lengend Snippet: Multiplex labeling efficiency. ( A ) and ( B ) Show the multiplex labeling efficiency calculated for different biomarkers from CT26 and 4T1 tumor images, respectively, where a global thresholding strategy was used to threshold for biomarker positivity. ( C ) and ( D ) Show the multiplex labeling efficiency calculated for different biomarkers from CT26 and 4T1 tumor images, respectively, where local thresholding was used to threshold for biomarker positivity.

Article Snippet: CT26, 4T1 and B16F10 cell lines were purchased from ATCC (Manassas, VA).

Techniques: Multiplex Assay, Labeling, Biomarker Discovery

Journal: Scientific Reports

Article Title: Quantitative performance assessment of Ultivue multiplex panels in formalin-fixed, paraffin-embedded human and murine tumor specimens

doi: 10.1038/s41598-024-58372-5

Figure Lengend Snippet: Reproducibility of cell–cell distance estimates in murine tumors. ( A ) Left image shows a hypothetical spatial distribution of two different cell types, i.e., A and B. The middle and right images show two scenarios where one of the cell types is the target and the other cell type is the reference. The arrows indicate the distance between a given reference cell and different target cells. Depending upon the distribution and abundance of the cell types, the choice of reference versus target will affect the numerical value of the cell–cell distance measurement. ( B ) and ( C ) Show the cell–cell distance estimates from CT26 and 4T1 tumor models, respectively, across all the runs. ( D ) Shows the CV of cell–cell distance estimates for CT26 and 4T1 tumor models. ( E ) Shows the average T cyt to T reg and T reg to T cyt distance estimates measured for the CT26 and 4T1 tumor models. **** p < 0.0001.

Article Snippet: CT26, 4T1 and B16F10 cell lines were purchased from ATCC (Manassas, VA).

Techniques: