Fig. 8: S5 was more efficient than IU1 in promoting autophagy.

a, b RAW264.7 cells were incubated with various concentrations of S5, IU1, or the same volume of DMSO in the absence or presence of LPS and IFN-γ at 10 ng/mL for 24 h. Cytotoxicity was detected by CCK-8 assay. Data are means ± SEM of five independent experiments. *P < 0.05 vs. DMSO group. c RAW264.7 cells were treated as in Fig. 7a, P62, LC3B, p-P65, P65, p-STAT1, and STAT1 expression were detected by western blot. Data are means ± SEM of five independent experiments. *P < 0.05, **P < 0.01 vs. LPS and IFN-γ group. d RAW264.7 cells were incubated with 5, 10, and 20 μM of IU1, co-immunoprecipitation experiments showed the K63-linked ubiquitination of Beclin1. e, f RAW264.7 cells were treated as above, and IL-6, TNF-α, and IL-1β protein level in medium were detected by ELISA assay (e) and mRNA expression were detected by RT-PCR (f). Data are means ± SEM of five independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. LPS and IFN-γ group. g RAW264.7 cells incubated with 20 μM of IU1 at 0, 2, 4, and 6 h, LC3B expression was analyzed by immunofluorescence staining. The average spot intensity in 1000 cells from each indicated sample was determined. Data are means ± SEM of five independent experiments. ***P < 0.001 vs. DMSO group. h RAW264.7 cells were incubated with various concentration of S5. The LC3 dots intensity was determined as in g.