Fig. 4
5-Nonyloxytryptamine (5-NL) and other inducers of CREB activation upregulate antigen presenting machinery in vitro and in vivo. A Protein expression of HTR1D in cancer cell lines was assessed using immunoblot analysis (a representative immunoblot of n = 3 is shown, cropping is indicated by a black frame). Lysates harvested from the mouse brain were used as a positive control. B Treatment with other HTR1D agonists for 18 h (Sumatriptan and L694247, both 3 μM) did not increase H2-Db/Kb protein expression in B16 cells as assessed using FACS (n = 3). C 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 12.5 mg/kg of Sumatriptan or with vehicle for five consecutive days. Tumor volume was measured (n = 6–7). (D, left panel) Representative immunofluorescent images of B16 cells treated with 5-NL (3 µM) or forskolin (10 µM) for 18 h and stained for phosphorylated CREB (p-CREB Ser-133) and H2-Db are shown (representative images of n = 3–4 are shown; scale bar indicates 50 µm) and fluorescent signal is quantified in D, right panel. E The adenylyl cyclase activator forskolin (20 μM) increased H2-Db/Kb protein expression in B16 cells following 18 h of treatment (n = 6) as measured by FACS. F B16 cells were treated with 5-NL and forskolin at the indicated doses for 72 h. Apoptosis was assessed using Annexin V/7AAD staining (n = 5). Percent apoptosis was ascertained by summing up the Annexin V+/7AAD− and Annexin V+/7AAD+ populations. G MC-38 cells were pre-treated for 30 min with the p-CREB inhibitor 3i (8 μM) followed by treatment with 3 µM of 5-NL for 24 h. Cells were analyzed using FACS for expression of H2-Db /Kb (n = 3–5). H C57BL/6 J mice were subcutaneously injected with 5 × 105 B16.GP33 or MC-38 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 13 days post tumor-inoculation and tumor tissue was stained for p-CREB using immunofluorescence. Scale bars indicate 50 µm and 20 µm (region of interest outlined in red) (representative images of tumors harvested from 4–6 mice are shown). Fluorescent signal from p-CREB channel was quantified in the right panel. 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 with a with a Dunnett’s post-hoc test