Fig 2: Adpa-Mn induces autophagic cell death. (A) HepG2 cells transfected with GFP-LC3 cDNA were treated with 20 µM Adpa-Mn for 12 h with or without pre-treatment with 5 mM 3-methyladenine (3-MA) for 2 h. The formation of vacuoles containing GFP-LC3 (dots) was examined by fluorescence microscopy. In another set of experiments, HepG2 cells were treated with 20 µM Adpa-Mn for 12 h with or without pre-treatment with 5 mM 3-MA for 2 h and then incubated with 0.05 mM monodansylcadaverine (MDC) for 10 min. The cells were then analyzed by fluorescence microscopy. Scale bar, 5 µm. Protein expression of LC3 in (B-D) was determined by western blot analysis. (B) HepG2 cells were cultured with 20 µM Adpa-Mn for 3, 6, 12, 24 and 48 h. (C) HepG2 cells were cultured with the indicated concentrations of Adpa-Mn for 24 h. (D) HepG2 cells were treated with 20 µM Adpa-Mn for 24 h with or without 3-MA, wartmanin and chloroquine (CQ) pre-treatment for 2 h. (E) HepG2 cells were treated with 20 µM Adpa-Mn for 24 h with or without 3-MA, and CQ pre-treatment for 2 h. MTT assay was used to evaluate the cell death rate. (F) HepG2 cells were transfected with control siRNA or siRNA targeting autophagy-related gene (ATG7). After 48 h, the cells were treated with 0, 5, 10 or 20 µM Adpa-Mn for 24 h, and cell death was measured by MTT assay. (G) The knockdown of ATG7 was confirmed by western blot analysis. Data represent the means ± SEM of 3 different experiments. *p<0.05 and **p<0.01 vs. respective control.

Fig 3: Inhibition of autophagy by knocking down Atg7 decreased ET release. NB4 cells were transiently transfected with Atg7 siRNA (siAtg7) at a concentration of 100 nM and scrambled siRNA (scr) was used as a negative control (siCT). Seventy-two hours after transfection, cells were treated with APL serum for 3 h. (a) The levels of LC3B-I and LC3B-II were detected by western blotting. (b) Quantification of the percentage of ET-releasing cells and the concentration of elastase–DNA complexes (n=5). All values are means±S.D. *P<0.05 versus control. (c) Immunofluorescence images showed inhibition of autophagy by knocking down Atg7 reduced the aggregation of LC3 (red) and ET formation (DAPI/Histone/LC3 merged). Bars represent 15 µm

Fig 4: The impact of siRNA ATG7 or bafilomycin A1 on OA-induced anti-cancer effect(A) T24 cells pre-transfected with adenovirus mCherry-GFP-LC3B were transfected with siRNA ATG7 or control siRNA for 72 h, then incubated with DMSO (v/v, 1:1000) or OA (7.5 µM) for another 24 h. Cells were then imaged by fluorescence microscopy. (B) CCK8 assay was used to analyze the viability of T24 cells. (C) Western blotting was used to analyze the protein level of ATG7, LC3B and Bcl-2. (D) T24 cells pre-transfected with adenovirus mCherry-GFP-LC3B were pre-treated with bafilomycin (100 nM) for 4 h, then incubated with DMSO (v/v, 1:1000) or OA (7.5 µM) for another 24 h. Cells were then imaged by fluorescence microscopy. (E) CCK8 assay was used to analyze the cell viability. (F) Western blotting was used to analyze the protein level of LC3B and Bcl-2. **P < 0.05. **P < 0.01.

Fig 5: Changes in detection of p62 and LC3 II/I protein upon induction or inhibition of autophagy. (A) In untreated wild type A549 cells, punctate GFP-LC3 (white arrow) was rare (left image) but increased significantly after exposure to rapamycin (10 µM/L) for 24 h (right image) by GFP-LC3 immunofluorescence microscopy. (B) Similar to wild type SPC-A-1 cells, punctate GFP-LC3 increased slightly after treatment with rapamycin. (C) Punctate GFP-LC3 positive cells were increased after rapamycin exposure both in A549 and SPC-A-1 cells. (D) p62 and LC3 II/I protein expression upon different concentrations of exposure to rapamycin for 24h. (E) Densitometric analysis of protein bands showing LC3 II/I protein expression increased from 5µM/L to 10µM/L and decreased from 50 µM/L to 100 µM/L, but p62 protein expression changes were not obvious. (F) Changes in detection of p62 and LC3 II/I protein upon autophagic gene 7 knockout (Atg7-/-). (G) Densitometric analysis of protein bands showing LC3 II/I protein expression almost disappeared after Atg7-/- knockout, but p62 protein expression decreased obviously both after autophagy induction by Rapamycin and autophagy inhibition by Atg7-/- knockout. Scale bar was 20 µm. wt, wild type A549 or SPC-A-1 cells; Rap, Rapamycin. *P < 0.05, **P < 0.01.