Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma

doi: 10.1186/s13046-024-03138-0

Figure Lengend Snippet: GLI1 regulates the expression of CX3CL1. A Representative cytokine array of the supernatants of B16F10 cells transduced with pBABE or pBABE-GLI1. B Expression level of cytokines that exhibited at least 1.5 fold change in pBABE-GLI1 compared to control. Protein expression was assessed by densitometric analysis using ImageJ, and each protein was normalized to array controls. The mean value of the proteins in pBABE was set to 1, and the fold change in pBABE-GLI1 was calculated for each protein. Data represent mean ± SEM. ** p < 0.01; *** p < 0.001 (unpaired Student t test). C, D Western blot of GLI1 in B16F10 and YUMM1.7 cells transduced as indicated. ACTIN was used as loading control. E, F qPCR of Cx3cl1 in B16F10 and YUMM1.7 cells transduced as indicated. Data represent mean ± SEM of three independent experiments. * p < 0.05; *** p < 0.001 (unpaired Student t test). G, H qPCR of GLI1 ( G ) and Cx3cl1 ( H ) in murine allografts injected with B16F10 cell transduced with pBABE or pBABE-GLI1. ** p < 0.01 (unpaired Student t test). I Western blot of GLI1 in YUMM5.2 cells transduced as indicated. ACTIN was used as loading control. J qPCR of Cx3cl1 in YUMM5.2 cells transduced as indicated. Data represent mean ± SEM of three independent experiments. * p < 0.05 (one-way ANOVA). K Western blot of GLI1 in human A375 melanoma cells transduced with pBABE or pBABE-GLI1. L qPCR analysis of CX3CL1 in A375 cells transduced with pBABE or pBABE-GLI1. Data represent mean ± SEM of three independent experiments. ** p < 0.01 (unpaired Student t test). M Schematic representation of the putative GLI consensus site in CX3CL1 promoter. N–O ChIP assay showing that GLI1 and RNA Pol II bind to CX3CL1 promoter in A375 cells. PTCH1 promoter was used as a positive control. The y axis represents the relative promoter enrichment, normalized on the input material. Data represent mean ± SD of three independent experiments. * p < 0.05; **** p < 0.0001 (unpaired Student t test)

Article Snippet: Conditioned media from 48 h incubation of sub-confluent cells in 2.5% FBS-containing media was applied to the Proteome Profiler Human XL Cytokine Array (cat# ARY022B, R&D System) or Proteome Profiler Mouse XL Cytokine Array (cat# ARY028, R&D System) following manufacturer’s instructions.

Techniques: Expressing, Transduction, Control, Western Blot, Injection, Positive Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma

doi: 10.1186/s13046-024-03138-0

Figure Lengend Snippet: CCL7 blockade reverts the activation of moDCs promoted by GLI1 silencing in melanoma cells. A Representative cytokine array in supernatants of SSM2c cells transduced with LV-c or LV-shGLI1. B Expression level of cytokines/chemokines exhibiting at least 1.5 fold change in LV-shGLI1 compared to control (LV-c), which was equated to 1. Protein expression was assessed by densitometric analysis using ImageJ and normalized to array controls. Data represent mean ± SEM. ** p < 0.01; *** p < 0.001 (unpaired Student t test). C Western blot of GLI1 in SSM2c and A375 transduced as indicated. ACTIN was used as loading control. D-E Validation of cytokine array with qPCR of genes shown in ( B ) in SSM2c ( C, D ) and A375 cells ( C, E ) transduced as indicated. Data are expressed as fold change relative to LV-c or pBABE, which were equated to 1. Gene expression is expressed as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. One-way ANOVA ( D ) and unpaired Student t test ( E ). F Representative immunofluorescence images of moDCs cultured 48 h in RPMI media (CTR), CM from SSM2c cells transduced with LV-c or LV-shGLI1 and treated with neutralizing CCL7 antibody or IgG isotype matched control. moDCs were stained with Phalloidin and counterstained with DAPI. Scale bar = 20 μm. G, H Cell circularity (4π*area/perimeter 2 ) ( G ) and cell perimeter (μm) ( H ) analysis of moDCs treated as indicated in ( F ) and quantified by ImageJ. Mean ± SEM from three donors of three independent experiments are reported. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (one-way ANOVA). I Schematic representation of moDC invasion assay. J Invasion assay of moDCs recruited after 48 h by CM from SSM2c cells transduced with LV-c or LV-shGLI1 and treated with blocking CCL7 antibody or IgG isotype. Data represents mean ± SEM of at least three independent experiments. ** p < 0.01; *** p < 0.001; ns, not significant (one-way ANOVA). K Representative pictures of ( J ). DAPI = 4’, 6-diamidino-2-phenylindole

Article Snippet: Conditioned media from 48 h incubation of sub-confluent cells in 2.5% FBS-containing media was applied to the Proteome Profiler Human XL Cytokine Array (cat# ARY022B, R&D System) or Proteome Profiler Mouse XL Cytokine Array (cat# ARY028, R&D System) following manufacturer’s instructions.

Techniques: Activation Assay, Transduction, Expressing, Control, Western Blot, Immunofluorescence, Cell Culture, Staining, Invasion Assay, Blocking Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma

doi: 10.1186/s13046-024-03138-0

Figure Lengend Snippet: An immune signature associated with high GLI and low cytokine expression predicts poor survival in patients with cutaneous melanoma. A Hierarchical clustering of cutaneous melanoma patients based on GLI and cytokine expression ( n = 443 patients, TCGA Pan-Cancer Atlas). B Pearson correlation of RNA expression of GLI1, GLI2 and cytokines CX3CL1, CXCL10, CCL7, CCL2, CXCL1, CCL20, and CXCL8 . C Kaplan–Meier survival curves for probability of survival comparing GLI High/Cytokine Low ( n = 274) (blue line) and GLI Low/Cytokine High ( n = 156) (red line) groups ( p = 0.018)

Article Snippet: Conditioned media from 48 h incubation of sub-confluent cells in 2.5% FBS-containing media was applied to the Proteome Profiler Human XL Cytokine Array (cat# ARY022B, R&D System) or Proteome Profiler Mouse XL Cytokine Array (cat# ARY028, R&D System) following manufacturer’s instructions.

Techniques: Expressing, RNA Expression

Journal: iScience

Article Title: Tumor-associated mesenchymal stromal cells modulate macrophage phagocytosis in stromal-rich colorectal cancer via PD-1 signaling

doi: 10.1016/j.isci.2024.110701

Figure Lengend Snippet: MSCs alter the secretory phenotype toward macrophage modulation and tumor promotion in a 3D CMS4-like culture system (A) Experimental outline depicting how the human 3D model of CMS4 CRC was generated by combining CRC cells, human bone marrow-derived MSCs and THP1 monocytes in a GelMA hydrogel for 10 days, with TNF-α addition at day 8 once complex spheroids had formed. (B) Representative scanning electron microscope image of a spheroid isolated from a triple cells 3D culture, scale bar = 50 μm. (C) Representative transmission electron microscope image of cells as part of the multicellular spheroid, scale bar = 2 μm. (D) Confocal microscopy images of Calcein AM (live) and Propidium Iodide (dead) stained gels, 10× magnification, of control and TNF-α treated samples. (E) Percentage cell viability of dissociated spheroids stained with Sytox blue and analyzed by flow cytometry ( n = 3) (top). Metabolic activity of the culture systems measured by Alamar blue and displayed as relative fluorescence at day 10 relative to day 1 ( n = 4) (bottom). (F) The secretome from the TNF-α treated 3D CRC culture system with or without MSCs was analyzed using a Proteome Profiler Human Cytokine Array Kit. Data from Human Cytokine arrays are expressed as pixel density of spots on the cytokine array membrane ( n = 2). (G) Proliferation of HCT116, hMSCs and primary monocytes in GelMA hydrogel +/− TNF-α for 10 days analyzed using CyQuant analysis kit. (H) The secretome from the TNF-α treated 3D CRC culture system with primary monocytes with or without MSCs was analyzed using a Proteome Profiler Human Cytokine Array Kit. Data from Human Cytokine arrays are expressed as pixel density of spots on the cytokine array membrane ( n = 2). Error bars, mean ± SD; ∗, p < 0.05, ∗∗, p < 0.01; ∗∗∗, p < 0.001 by two-way ANOVA and Tukey post hoc test (E) or paired t-test (F and H).

Article Snippet: The Proteome Profiler Human Cytokine Array Kit (ARY005B, R&D systems, Minnesota, United States) was used to assess the secretion of cytokines.

Techniques: Generated, Derivative Assay, Microscopy, Isolation, Transmission Assay, Confocal Microscopy, Staining, Control, Flow Cytometry, Activity Assay, Fluorescence, Membrane, CyQUANT Assay

Journal: iScience

Article Title: Tumor-associated mesenchymal stromal cells modulate macrophage phagocytosis in stromal-rich colorectal cancer via PD-1 signaling

doi: 10.1016/j.isci.2024.110701

Figure Lengend Snippet:

Article Snippet: The Proteome Profiler Human Cytokine Array Kit (ARY005B, R&D systems, Minnesota, United States) was used to assess the secretion of cytokines.

Techniques: Blocking Assay, Recombinant, Lysis, Saline, Flow Cytometry, Staining, Isolation, Viability Assay, CyQUANT Assay, Proliferation Assay, Software

Journal: Cell Death & Disease

Article Title: Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment

doi: 10.1038/cddis.2013.88

Figure Lengend Snippet: Characterization of HS-181 cell line after treatment with zebularine. ( a ) RT-PCR of cardiac markers after treatment with zebularine. Myh7, Myh6, Actc, cTnI and Serca2 present more expression after treatment. ( b ) Immunostaining of treated cells to detect cardiac-specific proteins. Scale bars; 50 μ m. ( c ) Blots of Proteome Profiler Array and the resulting quantification histograms demonstrating inhibition of pluripotency marker expression and ( d ) increased levels of mesodermic proteins after zebularine treatment (black arrows)

Article Snippet: Protein expression profiles were assayed using the specific human pluripotent Stem Cell array kit ‘Proteome Profiler Array' (R&D Systems Europe, Abingdon, UK; ARY010) following the manufacturer's instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Inhibition, Marker