Fig 2: Identification of Am-F4a as a novel ATG4B inhibitor.a Chemical structure of Am-F4a. b ATG4B (0.75 µg/mL) was incubated with or without Am-F4a (10 µM) at 37 °C for 30 min, FRET-GATE-16 was then added and incubated at 37 °C for another 30 min. The inhibitory effect of Am-F4a was detected by SDS-PAGE according to the cleavage of FRET substrates. FMK-9a was used as a positive control. c The inhibitory effect and IC50 of Am-F4a on ATG4B activity were obtained from FRET assay. d Surface plasmon resonance (SPR) analysis of binding affinity of Am-F4a to ATG4B. The Kd value was calculated based on the fitted curves. e Am-F4a on ATG4A activity, the inhibitory effect of Am-F4a was detected by FREAT. f, g The predicted 2D and 3D binding mode of Am-F4a with ATG4B by molecular docking. In 3D mode, the protein and ligand Am-F4a are shown by cartoon and stick respectively, the highlighted interacting residues are shown by lines, the hydrogen bonds are labeled by yellow dashed lines, and the hydrophobic interaction are labeled by gray dashed lines.

Fig 3: ATG4B plays an important role in ChCILpre-exos-mediated inhibition of autophagy.a RT-PCR was used to detect the mRNA change of autophagy-related gene in PMA-induced THP-1 cells with the indicated treatments. b PMA-induced THP-1 cells were given with the indicated treatments for 12 h and then the ATG4B protein level was detected by western blot. c PMA-induced THP-1 cells were preincubated with 10 µM CHX for the indicated time, and then incubated swChCNCpre-exos or swChCILpre-exos in the presence of LPS for an additional 12 h. Subsequently, the ATG4B protein level was evaluated by western blot. d PMA-induced THP-1 cells were transfected with Lenti-ATG4B shRNA or its control shRNA. Then the protein levels of ATG4B and LC3I/II were detected by western blot. e, f PMA-induced THP-1 cells were treated as d and then the immunofluorescence assay was taken to measure the LC3-puncta number per cell (e) and the ratio of ASC-specking cells (f). ANOVA with Bonferroni’s multiple comparison test was used, ***p < 0.001; *p < 0.05; ns, no significance

Fig 4: NR1 suppressed ACR-induced autophagy by regulating ITGAV in the hippocampus. (A) The expression of Trx-1 in the hippocampus was detected by western blot analysis. NR1 restored the expression of Trx-1. (B) The expression of ITGAV in the hippocampus was detected by western blot analysis. NR1 restored the expression of ITGAV. (C) The expression of ATG4B in the hippocampus was detected by western blot analysis. NR1 restored the expression of ATG4B. (D) The expression of LC3II in the hippocampus was detected by western blot analysis. NR1 restored the expression of LC3II. (E) The expression of Cathepsin D in the hippocampus was detected by western blot analysis. NR1 restored the expression of Cathepsin D. (F) The expression of LAMP2a in the hippocampus was detected by western blot analysis. NR1 restored the expression of LAMP2a. Each bar represents the mean ± SE (n = 6). n.s. (no significance) >0.05, *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant.

Fig 5: miR-449a-5p contributes to ChCILpre-exos-mediated decrease of ATG4B level.a Dio-labeled swChC-exos were coincubated with PMA-induced THP-1 cells and then the distribution of green fluorescence was observed under the fluorescence microscope. b The total RNA from pChCNCpre-exos or pChCILpre-exos were separately subjected to RNA sequencing analysis. The candidates from RNA-seq results and Bioinformatics analysis of ATG4B 3'UTR were listed. c PMA-induced THP-1 cells were transfected with the indicated miR-inhibitors, followed with pChCNCpre-exos or pChCILpre-exos in the presence of LPS. Then the ATG4B mRNA were detected by RT-PCR. d PMA-induced THP-1 cells were transfected with the inhibitor-miR-449a-5p and the negative control, followed with pChCNCpre-exos or pChCILpre-exos in the presence of LPS. Then the ATG4B protein level was detected by western blot. ANOVA with Bonferroni’s multiple comparison test was used, *p < 0.05; ns, no significance