Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteome and Secretome Profiling of the Melanoma-Induced Transition Toward Immune Incompetent Dendritic Cells Reveals Enhanced IDO1, Cathepsin, and Legumain Activity

doi: 10.1016/j.mcpro.2025.101048

Figure Lengend Snippet: Increased IDO activity in tumor-associated DC3s. A, kynurenine levels in 48 h supernatants of 50,000 dendritic cells (DCs) measured by HPLC, to analyze conversion of L-tryptophan to kynurenine as measure of indoleamine-2,3-dioxygenase (IDO) activity. DCs were cultured in 100 μl medium or medium with 50% BLM-CM, containing 10 mM L-tryptophan and increasing concentrations of the IDO inhibitor Epacadostat. Each circle represents an individual donor, bars represent mean values (N = 2 independent experiments, ratio paired t test between kynurenine levels of DC2 and ti-DC3, without inhibitor, results in p = 0.046). B, intracellular IDO levels of freshly isolated DCs from healthy donors, shown as log 2 fold change (FC) of CD1c + CD14 + DC3 versus CD1c + CD14 - DC2s. Each circle represents an individual donor (N = 4), bars are mean ± SD (Paired t test on raw values, ∗ p < 0.05). CM, conditioned medium; ti-DC3, tumor-induced DC3.

Article Snippet: PBMCs used for isolation of CD1c + CD14 − cells were depleted for CD3, CD56, CD19, and CD14 using MACS microbeads and LD columns prior to positive selection of CD1c using the CD1c (BDCA1) DC isolation kit (Miltenyi) following manufacturer's protocol.

Techniques: Activity Assay, Cell Culture, Isolation