Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Cytometry

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Splenic pan DCs were isolated from naïve BALB/c mice and analyzed by flow cytometry to characterize pre-cDC1s and CD8α+ cDC1s. (A) Representative flow plot demonstrating gating strategy. Gated on CD11c+B220- (conventional DCs); pre-cDC1s are gated as CD24 high CD8α- and CD8α+ cDC1s are gated as CD8α+. (B) Mean absolute cell number of respective DC subsets shown with SEM. (C-F) Mean percent and MFI with representative histograms shown with SEM for expression of (C) PD-L1, (D) PIR-B, (E) CD70, and (F) ICOSL on CD8α+ cDC1s (black) and pre-cDC1s (cyan). Data is representative of two experiments. (n = 5 per group). Paired t tests were used to determine significance between DC subsets. **P<0.01, ***P<0.001.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and analyzed by flow cytometry. Data is representative of three independent experiments. (n = 4 per group) (A) Representative flow plots depicting relative cDC1 proportions in naïve DCs (left) and 2.43 mAb DCs (right). Gated on CD11c+B220- (conventional DCs). (B) Mean percent of CD8α+ cDC1s (black) and pre-cDC1s (cyan) in untreated and 2.43 mAb-treated DCs shown with SEM. Two-way ANOVA and Dunnett’s multiple comparisons used to determine significance. **P<0.01, ****P<0.0001. (C) Mean absolute cell number of pre-cDC1s (CD24 high CD8α-) in untreated (black) and 2.43 mAb-treated (magenta) groups shown with SEM. Unpaired t test used to determine significance. *P<0.05. (D) Schematic depicting experimental design for proposed allogeneic mixed leukocyte reaction (MLR) using 2.43 mAb treatment.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Flow Cytometry

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and plated in an allogeneic MLR. Allogeneic T-cell proliferation was assessed on day 3 and day 4 of co-culture. Data is representative of two independent experiments. (n = 4 per group) (A-B) ModFit Software analysis of CellTrace dye dilutions. (A) Representative histograms depicting proliferation of H2K b + T-cells following stimulation with naïve DCs (top) or 2.43 mAb DCs (bottom) on days 3 and 4. Proliferation Index (PI) for representative histograms is boxed. (B) Mean PI for naïve or 2.43 mAb DCs shown with SEM. (C-G) Flow analysis of the proliferative fraction of T-cells was determined by first gating on H2K b +CellTrace low T-cells. (C) Mean percent of proliferated T-cells that are CD4+ (left) and CD8+ (right) on day 3 shown with SEM. (D) Mean percent of proliferated CD4+ (left) and CD8+ (right) T-cells expressing PD-1 on day 3 shown with SEM. (E) Mean percent of proliferated T-cells that are CD4+ (left) and CD8+ (right) on day 4 shown with SEM. (F) Mean percent of proliferated CD4+ (left) and CD8+ (right) T-cells expressing PD-1 on day 4 shown with SEM. (G) Mean percent of proliferated T-cells (left) and CD4+ T-cells (right) that are Propidium Iodide+ shown with SEM. Unpaired t tests were used to determine significance between groups. *P<0.05.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Co-Culture Assay, Software, Expressing

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: BALB/c mice were dosed with 200 μg of 2.43 mAb antibody intraperitoneally on day -3 and day -1. On day 0, splenic DCs were isolated and plated in an allogeneic MLR. DC composition was determined by flow cytometry on day 2 of co-culture. Data is representative of two independent experiments. (n = 4 per group) (A) Representative flow plots depicting percent of H2K d +CD11c+ DCs in naïve DCs (left) and 2.43 mAb DCs (right) on day 2 of co-culture. (B) Mean percent of H2K d +CD11c+ DCs for naïve (black) or 2.43 mAb DCs (magenta) shown with SEM. Unpaired t test was used to determine significance between groups. **P<0.01. (C) Representative flow plots depicting percent CD8α+ cDC1s and pre-cDC1s in naïve (left) and 2.43 mAb DCs (right). (D) Mean percent CD8α+ cDC1s (black bars) and pre-cDC1s (teal bars) in naïve and 2.43 mAb DCs shown with SEM. 2way ANOVA and Šidák’s multiple comparison used to determine statistical significance. **P<0.01.

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, Flow Cytometry, Co-Culture Assay, Comparison

Journal: PLoS ONE

Article Title: Murine precursors to type 1 conventional dendritic cells induce tumor cytotoxicity and exhibit activated PD-1/PD-L1 pathway

doi: 10.1371/journal.pone.0273075

Figure Lengend Snippet: Splenic DCs were isolated from naïve BALB/c mice and pre-cDC1s and CD8α+ cDC1s were cell sorted for RNA sequencing. (A) Enhanced volcano plot identifying genes that are up or down-regulated in pre-cDC1s compared to CD8α+ cDC1s. (B-C) Pathway results using Qiagen’s IPA software. P = 0.05 corresponds to a 1.3 -log p-value, and anything above this value is considered statistically significant. (B) Pathways predicted to be significantly inhibited (z-score < -2) in pre-cDC1s compared to CD8α+ cDC1s. (C) Pathways predicted to be significantly activated (z-score < 2) in pre-cDC1s compared to CD8α+ cDC1s. (D) Heat map showing relative gene expression levels for the PD-1/PD-L1 cancer immunotherapy pathway. Full documentation, methodology, and raw data for this data set is available online at: https://doi.org/10.25422/azu.data.14241902 .

Article Snippet: Anti-mouse CD8α PE-Cyanine7 , 53–6.7 , Thermo Fisher.

Techniques: Isolation, RNA Sequencing, Software, Gene Expression

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: Multiplex fluorescent immunostaining and 3D image reconstruction of whole core needle biopsies using T3. a Diagram shows slicing and imaging of cross-section of cores to assess antibody penetration after multiplex immunostaining. b Cross section images from BALB-NeuT tumor core after immunostaining for Her2 (green), CD3 (yellow), CD8 (red), CD31 (cyan), F4/80 (magenta), and ER-TR7 (gray). Left column shows 10X objective scan of 0.84 mm diameter core. Right column shows 40X objective region-of-interest imaging of the center of the cross section (white dotted square) demonstrating staining of tumor cells, T cells, microvasculature, macrophages, and fibroblasts. Scale bars: 100 μm (left) and 20 μm (right). c Six-plex immunostaining, clearing, scanning and image analysis of a whole BALB-NeuT tumor core (top image) reveals 3D distributions of Her2 tumor cell marker (green), CD3 T cell marker (yellow), CD8 cytotoxic T cell marker (red), CD31 endothelial marker (cyan), F4/80 macrophage marker (magenta), and ER-TR7 fibroblast marker (gray), displaying each channel in pseudocolor individually and in a merged image. Scale bar: 500 μm. d High resolution 2D images of the cell markers in the core. Scale bar: 10 μm.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques: Multiplex Assay, Immunostaining, Imaging, Staining, Marker

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: 3D visualization and mapping of CTLs in whole head and neck cancer cores. a Cores obtained with an 18 gauge automated device from head and neck cancer samples from patients 1 and 2 (left images) were immunostained for EGFR (green), CD3 (yellow), CD8 (red), and CD31 (cyan), cleared and scanned. 3D rendering of whole cores are shown for each channel and a merged image. Scale bar: 500 μm. b High resolution 3D view (left) and X-Y tomographic visualization (right) of a volume within the rendered core models, showing distributions of CD3 + CD8 + CTLs (orange) and CD31 + endothelium (cyan) within the EGFR + (green) tumor tissue and stroma. Scale bar: 30 μm. c Serial tomographic sections of X-Y planes at different Z-stack depths showing CTLs (orange) and endothelium (cyan) within the EGFR + (green) tumor tissue.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: 3D mapping and analysis of cytotoxic T lymphocytes in whole mouse core needle biopsy. a 3D spatial mapping of CD3 + CD8 + cytotoxic T lymphocytes (CTLs) in cores from six NeuT tumors displays local clustering and inhomogeneity. Scale bar: 500 μm. b Enumeration of CTLs in virtual optical sections along the long axis of each core in a demonstrates heterogeneous distribution of CTLs. c Conventional IHC after T3 confirms non-uniform distribution of CD8 + cells. Core #1 in a was formalin fixed and paraffin embedded, sectioned, immunostained, counterstained, and scanned. Inset displays high-magnification image of CD8 + cells. Scale bar: 1 mm (top) and 10 μm (inset). d Manual counting of CD8 + cells after IHC. Every seventh 5 μm serial section was stained and counted.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques: Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Non-destructive, multiplex three-dimensional mapping of immune infiltrates in core needle biopsy

doi: 10.1038/s41374-018-0156-y

Figure Lengend Snippet: Spatial correlation between CTLs and microvasculature in tumor parenchyma in head and neck cancer. a Relative quantification of EGFR + parenchyma (%) in sets of three cores each from six excised head and neck cancer patient samples. Data are expressed as mean ± SEM. b Quantification of relative volumes (%, mean ± SEM, n = 3) of CD3 + CD8 + CTLs within the EGFR + parenchyma (orange) and the whole cores (green) for each patient sample. c Quantification of relative volumes (%, mean ± SEM, n = 3) of CD31 + microvasculature within the EGFR + parenchyma (orange) and the whole cores (green) for each patient. d Plot comparing normalized densities of CD3 + CD8 + CTLs and CD31 + microvasculature in EGFR + parenchyma for each patient (% CD3 + CD8 + ( b ) or % CD31 + ( c ) within EGFR + volume/% EGFR + volume ( a ), mean ± SEM, n = 3). Three groups (blue, red, and green) were defined using K-means cluster analysis ( P <0.05 for both X and Y axes), distinguishing one patient, 1, with a “hot”, inflamed tumor and another patient, 2, with a “cold”, non-inflamed tumor. e High resolution 2D images of CD3 + CD8 + CTLs and CD31 + endothelium in EGFR + parenchyma and stroma in Patients 1 (“hot”) and 2 (“cold”) cores. Scale bar: 10 μm. f 3D distance profiles of CD3 + CD8 + CTLs away from CD31 + blood vessels in Patient 1 (“hot”) core. Inset shows a color distance map between blood vessel and CTL surfaces.

Article Snippet: Rat anti-mouse CD8α antibody (eBioscience, 4SM16, 1:200) was applied overnight at 4°C in a humidity chamber.

Techniques: Quantitative Proteomics

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Ascorbic acid improves the cognitive level of aged mice and increases the number of CD8 + cells in aged mice. A Trajectory tracking map of mouse movement in the open field test, illustrating time spent in the central zone (seconds), average speed during movement episodes (cm/s), and total distance traveled within the central area. B Statistical chart of New Object Cognition Index. C Flow cytometry was utilized to analyze immune cell populations, including CD3 + T, CD4 + T, CD8 + T, CD11b + , and B cells, in the whole blood of middle-aged mice assigned to either the control group or the AA group ( D ). E Statistical analysis of the flow cytometry data was presented in a histogram format, with significance levels denoted as follows: ns ( P > 0.05), * ( P < 0.05), ** ( P < 0.01), and *** ( P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: AA facilitates the early differentiation of T cells while concurrently suppressing myeloid differentiation. A A schematic depicting the early and long-term differentiation of T cells in an OP9-DL1 and lin-CD117 + HSC co-culture system is presented. B The diagram illustrates the progression of T cell maturation from CD44 and CD25 negative to positive selection. C Flow cytometry analysis and statistical bar graphs are utilized to demonstrate the AA-promoted changes in T cell DN stages, including the DN1 phase (CD44 + CD25 − ), DN2 phase (CD44 + CD25 + ), DN3 phase (CD44 − CD25 + ), and DN4 phase (CD44 − CD25 − ). D Flow cytometry analysis is utilized to examine the inhibition of myeloid differentiation, as well as to assess the long-term T cell differentiation promoted by AA in vitro ( E ). F and G Statistical bar graphs are generated to represent the flow cytometry results of CD8 + and CD4 + T cells, with significance levels denoted as ns: not significant ( P > 0.05), * ( P < 0.05), and ** ( P < 0.01), ( n = 3 for each group)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Co-Culture Assay, Selection, Flow Cytometry, Inhibition, Cell Differentiation, In Vitro, Generated

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: The Myh9 protein exhibits binding affinity towards AA and affects the proliferation of CD8 + T cells. A MetPro protein-metabolite interaction experimental workflow diagram. B Predicted binding differential protein KEGG pathway enrichment diagram. C Mass spectrometry predicted binding differential protein heatmap. D Protein interaction average degree analysis diagram. E Protein interaction network analysis diagram. F Concentration gradient binding curves of AA with Rhoa and Myh9 highlight the binding dynamics between these proteins. G Flow cytometry diagrams of CD8 + T cells for (Ga) control group, (Gb) AA group, (Gc) blebbistatin group, and (Gd) blebbistatin + AA group with statistical results ( H ) ( n = 3 for each group. ** p < 0.01, * p < 0.05). I Flow cytometry diagrams of CD11b + cells for (Ia) control group, (Ib) AA group, (Ic) blebbistatin group, and (Id) blebbistatin + AA group with statistical results ( J ) ( n = 3 for each group. **** p < 0.0001, *** p < 0.001, ** p < 0.01)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Binding Assay, Mass Spectrometry, Concentration Assay, Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Anti-CD8α antibody injection reduced the cognitive level of young mice. A Flow cytometry analysis of CD8 + T cells versus CD4 + T cells in the peripheral blood of mice after injection of IgG2b antibody and Anti-CD8α antibody ( B ). C Statistical histogram of CD8 + T flow cytometry results. D CD4 + T flow cytometry results statistical histogram. E Flow cytometry analysis of NK cells in mouse peripheral blood after IgG2b and Anti-CD8α antibody injection. F Statistical histogram of flow cytometry results of FNK cells. G Tracking diagram of mouse movement trajectory in open field test after antibody injection. H Time (s), speed, and distance traveled by the mouse in the central region during the open field test. I Statistical chart of new object cognition index ( n = 5 for each group, ns P > 0.05, * p < 0.05, *** P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Injection, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 is highly expressed in tumors and is associated with an immunosuppressive microenvironment. (A) Flowchart for screening key N6-methyladenosine (m6A) modification genes related to immunotherapy response in bladder cancer (BLCA). (B) Pearson correlation analysis bar chart of the 10 target genes with the percentage of complete response (CR) patients to immunotherapy in the IMvigor210 cohort, and a scatter plot of METTL3 expression level versus CR patient percentage. (C) Proportion of immunotherapy responses among different Lund subtypes in the IMvigor210 cohort. (D) Violin plot of METTL3 expression levels in bladder tissues of patients with different Lund subtypes. (E–F) Expression and statistical analysis of METTL3 in normal and tumor cells from single-cell sequencing of clinical bladder cancer samples. Histogram of METTL3 expression levels in cancer tissues versus adjacent normal tissues in (G) non-paired samples and (H) paired samples from the The Cancer Genome Atlas (TCGA) bladder cancer cohort. (I) Representative immunohistochemistry staining of METTL3 in clinical BLCA samples. (J–K) Scatter plots of METTL3 expression levels with CD8+T cell, cytotoxic cell, and myeloid-derived suppressor cell (MDSC) infiltration levels based on ssGSEA algorithm and TIMER V.2.0 database. (L) Statistical plot of METTL3 expression levels and immune scores in BLCA from the CAMOIP database. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Modification, Expressing, Sequencing, Immunohistochemistry, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 regulates bladder cancer progression by chemotactic CD8+T cell infiltration through the IGF2BP1-AHR-CCL5 axis. (A) Venn diagram illustrating the screening process for key transcription factors regulated by METTL3-mediated m6A modification and involved in CCL5 transcription. (B) Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of AHR and CCL5 mRNA expression levels after AHR knockdown in MB49 cells. (C) Assessment of CCL5 mRNA expression levels after overexpression of METTL3 and/or knockdown of AHR in MB49 cells. (D) Schematic representation of AHR binding sites within the CCL5 promoter region as predicted by JASPAR. (E) CHIP-qPCR analysis of AHR enrichment at the CCL5 promoter region. (F) mRNA and (G) protein expression levels of AHR after METTL3 knockdown in MB49 cells. (H) Peak plot of m6A modification sites in AHR in MB49 cells. (I) MeRIP-qPCR analysis showing changes in AHR m6A modification levels following METTL3 knockdown in MB49 cells. (J) RIP-qPCR analysis of METTL3 enrichment in AHR mRNA in MB49 cells. (K) MeRIP-qPCR showing changes in AHR m6A modification levels after treatment with the METTL3 inhibitor STM2457 in MB49 cells. (L) RT-qPCR analysis of AHR mRNA levels after STM2457 treatment to inhibit METTL3 in MB49 cells. (M) RNA decay assay showing AHR mRNA stability after silencing METTL3. (N) RNA decay assay showing AHR mRNA stability after treatment with METTL3 inhibitor STM2457 (2 µg/mL, 72 hours) in MB49 cells. (O) RT-qPCR analysis of IGF2BP1 and AHR mRNA expression levels in MB49 cells after silencing IGF2BP1. (P) RT-qPCR analysis of IGF2BP2 and AHR mRNA expression levels in MB49 cells after silencing IGF2BP2. (Q) RT-qPCR analysis of METTL3, IGF2BP1, and AHR mRNA expression levels in MB49 cells after overexpression of METTL3 and/or silencing of IGF2BP1. (R) Images of tumors formed by MB49 stable cell lines (control, AHR overexpression, METTL3 knockdown, METTL3 knockdown with AHR overexpression) subcutaneously implanted into the backs of C57BL/6J mice. (S) Growth curves of mouse bladder cancer tumors. (T) Volume of mouse bladder cancer tumors. (U) Schematic of the animal experiment. (V) Images of bladder cancer tumors in mice. (W) Growth curves of bladder cancer tumors in mice. (X) Tumor weights of bladder cancer tumors in mice; ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Modification, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Binding Assay, ChIP-qPCR, Stable Transfection, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: Targeting METTL3 enhances the efficacy of anti-Programmed Cell Death Protein 1 (PD-1) immunotherapy in bladder cancer. (A) Control and METTL3-knockdown MB49 stable cell lines were subcutaneously injected into mice. Anti-PD-1 antibody (200 µg/mouse, every 3 days) was administered intraperitoneally starting on day 6. Tumors were harvested on day 12 for flow cytometric analysis of the immune microenvironment (n=5). (B–D) Images, growth curves, and tumor weights of subcutaneous bladder cancer tumors in mice. (E–F) Flow cytometric analysis of MDSCs and CD8+T cell infiltration levels in the tumor tissues of mouse bladder cancer. (G) Wild-type MB49 cells were subcutaneously injected into mice, and on day 6, the mice were randomly divided into groups. Treatment included anti-PD-1 antibody (200 µg/mouse, every 3 days, intraperitoneally), IgG antibody (200 µg/mouse, every 3 days, intraperitoneally), the METTL3 inhibitor STM2457 (250 µg/tumor, once daily, intratumorally), and a combination of STM2457 and anti-PD-1 antibody. (H, J) Images, growth curves, and tumor weights of bladder cancer tumors in mice. (K) Control or METTL3 knockdown MB49 stable cell lines were orthotopically injected into the mouse bladder wall to establish an orthotopic bladder cancer model. Anti-PD-1 antibody (200 µg/mouse, every 3 days, intraperitoneally) or IgG antibody (200 µg/mouse, every 3 days, intraperitoneally) was administered starting on day 6 (n=5). (L) In vivo imaging system (IVIS) Living imaging of tumor growth in the orthotopic bladder cancer model. (M) Images of orthotopic bladder cancer tumors in mice. (N) Statistical analysis of fluorescence signal values from IVIS Living imaging on day 16. (O) Tumor volume in the orthotopic bladder cancer model. (P) Tumor weight in the orthotopic bladder cancer model. (Q) Schematic diagram of the study content. ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Control, Knockdown, Stable Transfection, Injection, In Vivo Imaging, Imaging, Fluorescence