Journal: Cell Reports Medicine

Article Title: Pan-cancer mapping of single CD8 + T cell profiles reveals a TCF1:CXCR6 axis regulating CD28 co-stimulation and anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101640

Figure Lengend Snippet: CXCR6 and CXCL16 expression in human and murine tumors tracks with dysfunctional T cells and myeloid cells, respectively (A) Mean expression (color bar) and fraction of expressing cells (dot size) for CXCR6 and CXCL16 (columns) across cell types (rows) in different human tumor studies (panels). (B) GSEA plots obtained for the KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION gene set when compared against the ranking of genes based on their co-expression with CXCL16 in macrophages in the indicated tumors. (C) Frequency of CXCR6 + cells (y axis, mean ± SEM) in the indicated subsets (x axis) of total (top) or Ova-Dextramer + (bottom) CD8 + TILs harvested from B16F10, B16Ova, or Mc38Ova hi tumors (top: B16F10, n = 8, 2 experiments combined. B16Ova, n = 10, 2 experiments combined. Mc38Ova hi , n = 9, 2 experiments combined. Bottom: B16Ova, n = 3, 1 experiment. Mc38Ova hi , n = 15, 2 experiments combined). (D) Representative distributions of expression levels (x axis, fluorescence intensity) in CXCR6 + and CXCR6 − CD8 + TILs from B16Ova tumors ( n = 4, 1 experiment). (E) Representative distributions of CXCL16 surface (top) or intracellular (bottom) expression (x axis, fluorescence intensity) in myeloid cells from Mc38Ova hi , B16Ova, or B16F10 tumors ( n = 4 per tumor, 1 experiment). (F) Left: B16Ova tumor area (y axis, mean ± SEM) of early-stage or late-stage tumors. Middle: frequency (y axis, mean ± SEM) of PD1- and TIM3-expressing CD8 + TILs (x axis) in early- or late-stage tumors. Right: frequency of CXCR6 + cells (y axis, mean ± SEM) in the indicated subsets (x axis) of CD8 + TILs from early- or late-stage B16Ova tumors ( n = 12, 2 experiments combined). Statistical significance was determined by Student’s unpaired t test (C, F). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-Mouse Tim3 APC Clone 5D12 , BD , Cat:# 567164 RRID: AB_2916481.

Techniques: Expressing, Fluorescence

Journal: Cell Reports Medicine

Article Title: Pan-cancer mapping of single CD8 + T cell profiles reveals a TCF1:CXCR6 axis regulating CD28 co-stimulation and anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101640

Figure Lengend Snippet: CXCR6 expression increases upon ICB and is repressed by TCF1 (A and B) Left: tumor area (y axis, mean ± SEM) over time (x axis) of Mc38Ova hi (A) or B16Ova (B) implanted in wild-type (WT) mice and treated with anti-PD1 (A), anti-PD-L1 + anti-TIM3 (B), or isotype control. Right: frequency of CXCR6 + cells (y axis, mean ± SEM) in the indicated subsets (x axis) of CD8 + TILs harvested from tumors treated as above (A: n = 4–6 per group, 1 experiment. B: n = 3–4 per group, 1 experiment). (C) Expression (row-normalized TPM, color bar) of top differentially expressed genes (rows) between WT OTI (E8i-Cre - , Tcf7 FL/FL ) and Tcf7 cKO OTI (E8i-Cre + , Tcf7 FL/FL ) cells at different time points after T cell activation (columns) ( n = 3, 1 experiment). (D) Mean expression (color bar) and fraction of expressing cells (dot size) of key genes (rows) in different CD8 + T cell clusters (columns) as determined by scRNA-seq of cells from B16Ova tumors implanted in WT or Tcf7 cKO mice ( n = 3 per group combined, 1 experiment). (E) Frequency of CXCR6 + cells (y axis, mean ± SEM) in the indicated subsets (x axis) of CD8 + TILs from B16Ova tumors from WT (black) or Tcf7 cKO (green) mice ( n = 3–7 per group, representative of 2 experiments). (F) Percent input (y axis, mean ± SEM) following chromatin immunoprecipitation (ChIP) PCR of the Cxcr6 locus with anti-TCF1 or rabbit IgG control antibodies (x axis) in WT or Tcf7 cKO CD8 + T cells ( n = 8, 5 experiments combined). (G–I) Luciferase activity (RLU, relative light unit, y axis, mean ± SEM) in HEK293T cells transfected with Cxcr6 locus-containing pGL4.10 luciferase reporters together with either empty vector (control) or vectors encoding the indicated transcription factors (x axis). Firefly luciferase activity is presented relative to constitutive Renilla luciferase activity ( n = 3, representative of 2 experiments). Statistical significance was determined by linear mixed model (A left, B left), Student’s unpaired t test (A right, B right, E, F comparing WT to Tcf7 cKO anti-TCF1 samples), Student’s paired t test (F comparing WT samples), or one-way ANOVA with Tukey’s multiple comparisons test (G, H, I). NS = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-Mouse Tim3 APC Clone 5D12 , BD , Cat:# 567164 RRID: AB_2916481.

Techniques: Expressing, Control, Activation Assay, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Cell Reports Medicine

Article Title: Pan-cancer mapping of single CD8 + T cell profiles reveals a TCF1:CXCR6 axis regulating CD28 co-stimulation and anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101640

Figure Lengend Snippet:

Article Snippet: Anti-Mouse Tim3 APC Clone 5D12 , BD , Cat:# 567164 RRID: AB_2916481.

Techniques: Virus, Recombinant, Purification, SYBR Green Assay, Staining, Reporter Assay, In Vitro, Activation Assay, Generated, Luciferase, Plasmid Preparation, Sequencing, Software, CRISPR

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Ischemia-reperfusion injury elicits time-dependent changes of TECs injury, inflammatory infiltration, and interstitial fibrotic remodeling. A , schematic illustration of the experimental protocol for inducing renal IRI in a mouse model. B , levels of Scr from sham-operated mice and mice sacrificed at 1 h, 1 day, 3 days, 7 days, 28 days after I/R. C and D , protein expression profiling by western blot ( C ) and subsequent statistical analysis ( D ) of Havcr1/Kim1 in renal cortical samples. E , immunohistochemical detection of Havcr1/Kim1 expression in renal cortical sections from sham and I/R-treated mice. The scale bar represents 100 μm. F and G , protein expression profiling by western blot ( F ) and subsequent statistical analysis ( G ) of IL-6 in renal cortical samples. H , immunofluorescent staining for the macrophage marker F4/80 in renal cortical sections. The scale bar represents 40 μm. I , histopathological assessment of renal tissue architecture (H&E) and collagen deposition (Masson’s trichrome) in experimental groups. The scale bar represents 100 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Havcr, hepatitis A virus cellular receptor 1; IL-6, interleukin-6; IRI, ischemia-reperfusion injury; Kim1, kidney injury molecule-1; Scr, serum creatinine; TEC, tubular epithelial cell.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Marker, Virus

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Dock family proteins are identified, with knockdown of Dock2 demonstrating a significant attenuation of the proinflammatory response of TECs in vitro . A , heatmap of Dock family proteins and phosphosites (ANOVA, p < 0.05). B , intensity plots showing Dock2 ( black line ) and its phosphosite S1704 (Dock2_pS1704, green line ) across the six experimental groups. C and D , protein expression profiling by western blot ( C ) and subsequent statistical analysis ( D ) of Dock2 in renal cortical samples. E , immunofluorescent detection of Dock2 and Havcr1/Kim1 expression and colocalization in renal tissue sections from sham and I/R-treated mice. The scale bar represents 40 μm. F , the knockdown efficiency of Dock2 by siRNA in H/R-treated HK-2 cells was analyzed by western blot. G , the statistical analysis of ( F ). H – J , RT-qPCR showing the effects of Dock2 knockdown on mRNA expression levels of MCP-1, TNF-α, and IL-6. K , western blot analysis was performed to assess the effects of Dock2 knockdown on IKK-β phosphorylation in H/R-treated HK-2 cells. L , densitometric analysis of p-IKKβ from ( K ) was performed with normalization to the respective total protein. M , translocation of NF-κB p65 in HK-2 cells was detected by immunofluorescence. NF-κB p65 ( green ), DAPI ( blue ). The scale bar represents 5 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; Dock2, dedicator of cytokinesis 2; Havcr, hepatitis A virus cellular receptor 1; H/R, hypoxia/reoxygenation; IKKβ, IκB kinase beta; Kim1, kidney injury molecule-1; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; RT-qPCR, reverse transcription quantitative PCR; TEC, tubular epithelial cell; TNF-α, tumor necrosis factor-α.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Knockdown, In Vitro, Phospho-proteomics, Expressing, Western Blot, Quantitative RT-PCR, Translocation Assay, Immunofluorescence, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Inhibition of Dock2 by CPYPP attenuates tubular injury, inflammatory infiltration and interstitial fibrosis after renal IRI in mice. A , schematic of the experimental design for unilateral I/R in mice treated with CPYPP or vehicle via i.p. injection following I/R induction. B , measurement of Scr levels. C , H&E staining was performed to assess the effect of pharmacological inhibition of Dock2 by CPYPP on renal tissue architecture in mice at 3 days and 28 days post-I/R. Inflammatory cells, tubular casts, and dilated tubules are indicated by solid arrows , hollow arrows , and triangles , respectively. The scale bar represents 100 μm. D , tubular injury score at 3 days post-I/R (quantified from C ). E – I , western blot ( E ) and quantitative analysis ( F – I ) of renal cortical Havcr1/Kim1, IL-6, TNF-α, and MCP-1 expression in sham-operated and 3 days post-I/R mice, with or without CPYPP treatment. J , immunofluorescent staining for the macrophage marker F4/80 in renal cortical sections to assess inflammatory infiltration. The scale bar represents 40 μm. K , the quantitative analysis of the F4/80 immunofluorescence staining from ( J ). L – N , western blot ( L ) and quantitative analysis ( M and N ) of renal cortical FN and α-SMA expression in sham-operated and 28 days post-I/R mice, with or without CPYPP treatment. O , Masson's trichrome staining was used to evaluate the effect of CPYPP on renal collagen deposition at 28 days post-I/R, with quantification data shown in ( P ). The scale bar represents 100 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. CPYPP, 4-[3′-(2″-chlorophenyl)-2′-propen-1′-ylidene]-1-phenyl-3,5-pyrazolidinedione; Dock2, dedicator of cytokinesis 2; FN, fibronectin; Havcr, hepatitis A virus cellular receptor 1; IL-6, interleukin-6; IRI, ischemia-reperfusion injury; Kim1, kidney injury molecule-1; MCP-1, monocyte chemoattractant protein-1; α-SMA, alpha-smooth muscle actin; Scr, serum creatinine; TNF-α, tumor necrosis factor-α.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Inhibition, Injection, Staining, Western Blot, Expressing, Marker, Immunofluorescence, Virus