Fig. 5

Effect of autophagy modulators on cytokine production by FaDu cells. (a) Conditioned media (CM1 and CM2) preparation scheme. In CM1, everything except large EVs (up to 220 nm) and apoptotic bodies can be presented. CM1 involves free cytokines + cytokines in small EVs. CM2 was prepared from CM1. CM1 was concentrated and all molecules smaller than 100 kDa were removed (to remove free cytokines). (b) Heatmap of normalized cytokine levels in CM1. IL-6 and IL-8 respond similarly to autophagy modulation. Their expression is most promoted due to autophagy inhibition by EACC and, conversely, starvation suppresses their expression. (c) Heatmap of normalized cytokine levels in CM2. The biggest changes were seen in the levels of IL-8. (d) Cytokines detected in PS-EVs during proteomic analysis. PG = protein group (e) and (f) proteins involved in cytokine production were significantly changed in PS-EVs after treatment with autophagy modulators. BAFA1 = bafilomycin, HCQ = 3-hydroxychloroquine, RAPA = rapamycin, BEZ = NVP-BEZ235, starv = starvation, APB = autophinib