Fig 2: AhR-overexpression decreased autophagy in CL1-5 cells.CL1-5 cells were transfected with pcDNA3.1-AhR for 24 h and autophagy-related proteins were then analysed by western blotting. (a) Western blotting showed that AhR overexpression decreased the levels of BNIP3 and LC3II/LC3I ratio. (b) BafA1 (5 nM) treatment of wild-type and AhR-overexpressing cells to compare autophagy flux. Quantification of the western blot data showing the fold-changes in BNIP3, p62, ATG12-5, LC3, and ATG7 expression. Full-length blots are presented in Supplementary Fig. S16. The data were analysed and expressed as the mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the control group. ###P < 0.001 indicate a statistical difference between wild-type and AhR overexpression cells in BafA1 treatment groups.

Fig 3: Level of autophagy-related protein expression in different cells was correlated with their cell motility.Protein expression was evaluated by western blotting. Cells (104 cells/Transwell) were seeded on matrigel-coated Transwell inserts, and incubated for 16 h. The migrated cells were stained with crystal violet and images were acquired at 40× magnification. (a) Protein expression of AhR, BNIP3, LC3, and ATG12-5 in A549, H1299, and CL1-5 cells. Full-length blots are presented in Supplementary Fig. S13. (b) Cell migration potential analysed by BafA1 treatment (5 nM) compared to the control among A549, H1299, and CL1-5 cells. (c) Protein expression of AhR, E-cadherin, and vimentin by BafA1 (5 nM) treatment of A549, H1299, and CL1-5 cells. Full-length blots are presented in Supplementary Fig. S14. (d) Immunofluorescence microscopy of LC3B (green) in A549 and CL1-5 cells following transfection with shAhR and pcDNA3.1-AhR vector. Nuclei were visualized by staining with Hoechst 33258 (blue). The number of puncta were quantified by Image ProTM, data were presented as percentages normalized to the control (100%). (e) Multifocal, well-demarcated, metastatic foci (arrow) were detected in the lung, there were no tumour detected in AhR and AhR + BafA1 groups. The metastatic tumour cells grew in a nest or sheet pattern and showed areas of glandular differentiation and papillary architecture (HE staining). The quantified data were analysed and expressed as the mean ± SD from three independent experiments. **P < 0.01; ***P < 0.001 compared to the control group.

Fig 4: IPMK Enhances Transcription of Autophagy-Related Genes(A) qPCR analysis of LC3B, BNIP3, BNIP3L, p62, GABARAPL1, and ATG12 in IPMK WT and KO MEFs after GluStv.(B) Western blot of BNIP3L, ATG12, and GABARAPL1 in IPMK WT and KO MEFs after GluStv.(C) Western blot of BNIP3L, ATG12, and GABARAPL1 in F/F and Cre (IPMK KO) mouse livers after 24 h of food starvation.(D) IPMK KO MEFs were stably transfected with empty vector (myc), wIPMK myc, and kinase-dead myc (KSA) IPMK. Shown is a qPCR analysis of LC3B, BNIP3, BNIP3L, p62, GABARAPL1, and ATG12 in myc, wIPMK, and KSA MEFs.(E) Western blot analysis of histone 4 lysine 16 acetylation (H4k16ac).(F) Chromatin immunoprecipitation of H4k16 at the LC3B promoter from WT and KO MEFs. n = 3, ***p < 0.001.(G) Immunoprecipitation of Sirt-1 and western blot of DBC1 before and after GluStv in WT and KO MEFs.(H) HEK293 cells were transfected with FLAG Sirt-1 and myc AMPK or empty vector of myc. Immunoprecipitation of myc was followed by FLAG western blotting.(I) HEK293 cells were transfected with FLAG Sirt-1 and myc IPMK and empty vector of myc. Immunoprecipitation of myc was followed by FLAG western blotting.Data are means ± SD.