Fig. 3
5-Nonyloxytryptamine (5-NL) upregulates antigen presenting machinery in human and murine tumors in vitro and in vivo. A Basal expression levels of H2-Db and H2-Kb in mouse cells and HLA A-C in human cells were assessed using flow cytometry (n = 5). B-D C57BL/6 J mice were subcutaneously injected with 5 × 105 B16.GP33 cells. 7 days post-tumor injection mice were randomized into two groups and treated daily with 6.25 mg/kg of 5-NL or with vehicle for five consecutive days. Mice were sacrificed on day 13 post tumor-inoculation. B Tumor sections were stained for MHC-I using immunofluorescence (representative images of tumors harvested from 4 mice are shown; scale bar indicates 50 µm). C H2-Db/Kb and PD-L1 protein expression on CD45.2− and (D) H2-Db/Kb expression on tumor infiltrating CD8+ and CD4+ T cells, Treg’s (CD4+CD25+FOXP3+), monocytes (CD11b+Ly6ChighLy6G−), granulocytes (CD11b+Ly6GhighLy6Clow), TAMs (CD11b+F4/80highLy6ClowLy6G−) and DCs (CD11c+MHC-II+) was assessed using flow cytometry (n = 5–16). E H2DB and H2KB mRNA expression in B16 cells treated with 5-NL for 18 h was assessed using RT-PCR. Expression was normalized to β-Actin (n = 4). F H2-Db/Kb and PD-L1 protein expression was assessed by flow cytometry following treatment with 5-NL for 18 h in B16 cells (phenotype was recapitulated in B16.GP33 cells, data not shown) (n = 5). G H2-Db/Kb (mouse cell lines) and HLA A-C (human cell lines) protein expression was assessed using flow cytometry following treatment with 5-NL (5 μM for RPMI-7591 and 3 μM for MC-38, SW620, A549, MDA-MB-231 and MOPC cells) for 24 h (n = 5–9). Error bars indicate SEM; *P < 0.05 as determined by a Student´s t-test (unpaired, 2 tailed) or a one-way ANOVA with a Tukey’s post-hoc test