melanoma cell lines murine b16 f10 melanoma cells  (ATCC)

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: (A) Oncoprint depicting the frequency and types of alterations of the NRP1 gene in 212 melanoma cancer and 2683 pan-cancer patient samples from the cBioPortal database. (B) Bar graph representation of alteration frequency of NRP1 gene in 2683 cancer patient samples from cBioPortal database. (C) Fold change in mRNA expression of NRP1 gene when compared between tumorigenic B16-F10 and non-tumorigenic NIH-3T3 cells. (D) Bar graphs representing apoptosis in tumorigenic B16-F10 and non-tumorigenic NIH-3T3 cells upon treatment with different concentrations of NRP1 inhibitor EG00229, which is determined by Annexin & 7-AAD dual staining. Representative bar diagrams are of three independent experiments. ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: (A) Flow cytometric dot plots showing alterations in the frequency of FOXP3, NKG2A and NRP1 positive purified splenic T cell population with or without anti-CD3/CD28 antibodies and B16-F10-CS treatment. Bar graph representation of alteration in frequency of (B) FOXP3, (C) NKG2A and (D) NRP1 positive purified splenic T cell population with or without anti-CD3/CD28 antibodies and B16-F10-CS treatment. Representative bar diagrams are of three independent experiments. ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Purification

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: (A) Flowcytometric histogram plots depicting alterations in the expression of NRP1, NKG2A and FOXP3 in anti-CD3/CD28 antibodies and B16-F10-CS treated purified splenic T cell with or without NRP1 inhibition. Bar graph representation of alteration in expression of (B) NRP1, (C) NKG2A and (D) FOXP3 in anti-CD3/CD28 antibodies and B16-F10-CS treated purified splenic T cell with or without NRP1 inhibition. Representative bar diagrams are of three independent experiments. ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Expressing, Purification, Inhibition

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: Bar graphs depicting the alteration in (A) TNF (B) IFN-γ, (C) IL-10, (D) IL-2, (E) IL-17(A), and (F) IL-6 secretion levels in anti-CD3/CD28 antibodies and B16-F10-CS treated purified splenic T cell with or without NRP1 inhibition. (G) Bar graph depicting proliferation of T cells via CFSE proliferation assay upon treatment with anti-CD3/CD28 antibodies and B16-F10-CS with or without NRP1 inhibition. (H) Bar graph depicting proliferation of effector T cells co-cultured with purified splenic T cells treated with anti-CD3/CD28 antibodies and B16-F10-CS with or without NRP1 inhibition. Representative bar diagrams are of three independent experiments. ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Purification, Inhibition, Proliferation Assay, Cell Culture

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: (A) Western blots showing expression of phosphorylated STAT3, STAT5, STAT1, SMAD2/3, PI3K, AKT, SAPK/JNK, P38 and ERK in anti-CD3/CD28 antibodies and B16-F10-CS treated purified splenic T cell with or without NRP1 inhibition. Representative bar graphs are of three independent experiments, showing relative expression levels of (B) p-STAT3, (C) p-STAT5, (D) p-STAT1, (E) p-SMAD2/3, (F) p-PI3K, (G) p-AKT and (H) p-ERK when compared with expression of total STAT3, STAT5, STAT1, SMAD2/3, PI3K, AKT and ERK respectively. Β-actin was used as a loading control for this experiment. ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Western Blot, Expressing, Purification, Inhibition, Control

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: Bar graphs depicting the alteration in expression of (A) NRP1, (B) NKG2A, (C) FOXP3, (D) T-bet, (E) GATA-3, (F) CTLA-4, (G) PD-1 and (H) PD-L1 in purified splenic T cells isolated from control healthy and B16-F10 subcutaneous tumour bearing C57BL/6 mice with or without NRP1 inhibitor treatment. Representative bar diagrams are of three independent experiments. ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Expressing, Purification, Isolation, Control

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: (A) Flowcytometric histogram plots depicting proliferation of purified splenic T cells isolated from B16-F10 subcutaneous tumour-bearing mice with or without treatment with Neuropillin-1 (NRP1) inhibitor. (B) Flowcytometric histogram plots depicting proliferation of effector T cells cultured with purified T cells isolated from B16-F10 subcutaneous tumour-bearing mice with or without NRP1 inhibitor treatment.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Purification, Isolation, Cell Culture

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: (A) Representative lung images of healthy control mice and lungs extracted from untreated and NRP1-treated C57BL/6 mice previously injected with B16-F10 cells intravenously. (B) Kaplan-Meier plot depicting the probability of survival of C57BL/6 mice with or without NRP1 inhibitor treatment after inoculation of 0.1X10 6 B16-F10 cells intravenously. (C) Bar graph depicting the number of metastatic nodes in the lungs of control healthy mice and in lungs extracted from untreated and NRP1 treated C57BL/6 mice previously injected with B16-F10 cells intravenously. (D) Kaplan-Meier plot depicting the probability of survival of C57BL/6 mice with or without NRP1 inhibitor treatment after inoculation of 0.3X10 6 B16-F10 cells subcutaneously. (E) XY plot depicting the volume of subcutaneous tumours in C57BL/6 mice with or without NRP1 inhibitor treatment after inoculation of 0.3X10 6 B16-F10 cells subcutaneously.

Article Snippet: The murine melanoma cell line B16-F10 (ATCC™ CRL-6475™) and the murine fibroblast cell line NIH-3T3 (ATCC™ CRL-1658™) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS; Gibco), 1X antibiotic solution (100X Liquid, HiMedia Laboratories Pvt.

Techniques: Control, Injection

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 expression in melanoma cells sustains expansion and recruitment of PMN-MDSCs in vitro. A Flow cytometry analysis of activated T cells and PMN-MDSCs co-cultures for 48 h at different ratios. The percentage of dead cells was determined by Viobility staining gated on CD3 + cells. Data represent mean ± SD of three independent experiments. ** p < 0.01; *** p < 0.001; ns, not significant (one-way ANOVA). B Western blot of GLI1 in B16F10 and YUMM1.7 murine melanoma cells transduced with pBABE or pBABE-GLI1. ACTIN was used as loading control. C, D Proliferation assay in PMN-MDSCs cultured with CM for 72 h from B16F10 ( C ) and YUMM1.7 ( D ) cells transduced as indicated. Data represent mean ± SEM of at least three independent experiments. *** p < 0.001; **** p < 0.0001 (unpaired Student t test). E Schematic representation of PMN-MDSC invasion assay. PMN-MDSCs were seeded in the upper chamber and CM from B16F10 and YUMM1.7 cells transduced with pBABE or pBABE-GLI1 in the lower chamber. The number of invading cells was counted after 72 h. F-I Invasion assay in PMN-MDSCs recruited by CM from B16F10 ( F, G ) and YUMM1.7 ( H, I ) transduced as indicated. Data represent mean ± SEM of at least three independent experiments. *** p < 0.001 (unpaired Student t test). ( G ) and ( I ) are representative pictures of ( F ) and ( H ) respectively. J Schematic representation of PMN-MDSC culture conditions for cytokines and adenosine determination. PMN-MDSCs were first conditioned with CM from B16F10 cells transduced with pBABE or pBABE-GLI1 for 48 h. After refreshing, PMN-MDSCs were cultured for 24 h with their media and supernatants collected for Milliplex analysis and adenosine quantification. K-N Concentration (pg/mL) of cytokines IL-6 ( K ), IL-1β ( L ), TGFβ1 ( M ) and CCL4 ( N ) measured by Luminex assay in supernatants of PMN-MDSCs treated as indicated. O Quantification of adenosine (μM) in PMN-MDSC supernatants by fluorometric assay. Data represent mean ± SD of at least three independent experiments * p < 0.05; ** p < 0.01 (unpaired Student t test)

Article Snippet: Murine melanoma cell lines YUMM1.7 and YUMM5.2 were purchased from LGC Standards ( www.lgcstandards.com ) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Euroclone, Milan, Italy) containing 10% fetal bovine serum (FBS) (Euroclone), 1% penicillin–streptomycin (P/S) solution (Thermo Fisher Scientific, Waltham, MA, USA) and 1% non-essential amino acids (Euroclone) at 37°C in a 5% CO 2 incubator.

Techniques: Expressing, In Vitro, Flow Cytometry, Staining, Western Blot, Transduction, Control, Proliferation Assay, Cell Culture, Invasion Assay, Concentration Assay, Luminex

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: Murine melanoma cell lines YUMM1.7 and YUMM5.2 were purchased from LGC Standards ( www.lgcstandards.com ) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Euroclone, Milan, Italy) containing 10% fetal bovine serum (FBS) (Euroclone), 1% penicillin–streptomycin (P/S) solution (Thermo Fisher Scientific, Waltham, MA, USA) and 1% non-essential amino acids (Euroclone) at 37°C in a 5% CO 2 incubator.

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: Blockage of CX3CL1 impairs GLI1-dependent expansion and invasion of PMN-MDSCs. A Schematic representation of PMN-MDSC culture conditioning with CM from B16F10 and YUMM1.7 cells transduced with pBABE or pBABE-GLI1 and treated with IgG or neutralizing CX3CL1 antibody (1.5 μg/mL). B, C Proliferation assay in PMN-MDSCs cultured 72 h with CM from B16F10 ( B ) or YUMM1.7 ( C ) cells transduced with pBABE or pBABE-GLI1 and treated with IgG or CX3CL1 antibody. D Schematic representation of MDSC invasion assay. PMN-MDSC were seeded in the upper chamber and CM from B16F10 and YUMM1.7 transduced with pBABE or pBABE-GLI1 and treated with IgG or CX3CL1 antibody in the lower chamber. The number of invading cells was counted after 72 h. E, F Invasion assay in PMN-MDSCs recruited after 72 h by CM from B16F10 ( E ) and YUMM1.7 ( F ) cells transduced with pBABE or pBABE-GLI1 and treated with IgG or CX3CL1 antibody. Data represent mean ± SD of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant (one-way ANOVA)

Article Snippet: Murine melanoma cell lines YUMM1.7 and YUMM5.2 were purchased from LGC Standards ( www.lgcstandards.com ) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Euroclone, Milan, Italy) containing 10% fetal bovine serum (FBS) (Euroclone), 1% penicillin–streptomycin (P/S) solution (Thermo Fisher Scientific, Waltham, MA, USA) and 1% non-essential amino acids (Euroclone) at 37°C in a 5% CO 2 incubator.

Techniques: Transduction, Proliferation Assay, Cell Culture, Invasion Assay