Figure 4.
Decreased suppressive activity of the A375 melanoma culture supernatants by pretreatment with RNAi for BRAFV600E alone, STAT3 alone, or both on LPS-induced IL-12 and TNF-α production from DCs. (a) Neutralization of IL-6, IL-10, or VEGF in the culture supernatants of A375 melanoma cells restored the inhibition of IL-12 production from LPS-stimulated human DCs. Human MoDCs were cultured with the A375 supernatants in the presence or absence of the monoclonal antibody for IL-6, IL-10, VEGF, or an isotype control antibody at 1 μg/ml. IL-12 production in the culture supernatants was determined by ELISA 24 h after the LPS stimulation at 100 ng/ml. (b) CD14+ monocytes were isolated from PBMCs by using MACS and cultured in the media containing RPMI 1640, 10% (vol/vol) FBS, 100 ng/ml GM-CSF, and 50 ng/ml IL-4 with or without 20% (vol/vol) of the culture supernatant of the A375mel cells prepared in Fig. 3. (a) Half of the media, cytokines, and the culture supernatant was changed every 2 d. On day 5 of the culture, LPS was added at 100 ng/ml. After 15 h, the culture supernatants were collected and subjected to ELISA of IL-12 and TNF-α.