Fig 2: Effects of circDHX8, ATG2B, and RNF5 on the growth and aggressiveness of GC cells.A Cell counting kit-8 analysis of cell proliferation in MKN45 transfected with corresponding plasmids. B Colony-forming capacity of MKN45 transfected with corresponding plasmids. C, D Transwell assays for determining the migration and invasion abilities of MKN45 cells transfected with corresponding plasmids. E A wound healing assay was performed to evaluate the migration ability of MKN45 cells transfected with corresponding plasmids. F Immunofluorescence staining of autophagosomes and autolysosomes in MKN45 cells transfected with corresponding plasmids. **P < 0.01 and ***P < 0.001.

Fig 3: Identification of sRNF5 as a sABHD16A-interacting protein. (A) The interaction between human ABHD16A and RNF5 was confirmed by Co-IP assay in HEK293 cells. Expression constructs of Flag-hRNF5 with empty vector or hABHD16A-EGFP were transfected into HEK293 cells for 24 h, and cell lysates were immunoprecipitated through the Flag epitope followed by immunoblotting using anti-GFP and anti-Flag antibody. (B) The topologies of Sus scrofa RNF5 (Red) and ABHD16A (Blue) were predicted by AlphaFold2. (C) The interacting interface and residues between sRNF5 (green) with sABHD16A (yellow) were shown. (D) The transmembrane regions of Sus scrofa RNF5 and ABHD16A were predicted by Phyre2 using “normal mode.” (E) sRNF5 interacted with sABHD16A through its transmembrane domain. PK15 cells were cotransfected with indicated plasmids for 24 h, and cell lysates were immunoprecipitated (IP) with anti-Flag antibody. Expression of protein was analyzed by immunoblotting with Flag and green fluorescent protein (GFP) antibody. ΔTM, Δ161–177 aa of sRNF5. (F) The gray value of immunoprecipitated sABHD16A-EGFP bands to GAPDH internal reference band was normalized. Data are mean ± SD from three independent experiments. ***P < 0.001 (unpaired two-tailed t test). (G) The interaction between endogenous sRNF5 and sABHD16A was confirmed by Co-IP assay in PK15 cells; IgG and anti-RNF5 were used as negative control and bait antibody for IP, respectively. (H) The schematic diagram of bimolecular fluorescence complementation assay (BiFC) assay. (I) Detection of sRNF5 and sABHD16A interaction in living cells by using BiFC assay. PK15 cells were transiently transfected with indicated plasmids. Twenty-four hours later, cell nucleus were stained with Hoechst 33342 and subjected to confocal microscopy. yellow fluorescent protein (YFP) signals denote the interaction in living cells. Scale bar, 20 µm. YN, 1 to 155 aa of YFP; YC, 156 to 239 aa of YFP. (J) Representative confocal images show colocalization between sRNF5 and sABHD16A on ER. PK15 cells were transfected with sABHD16A-DsRed. Twenty-four hours after transfection, cells were stained with ER Tracker Green for 0.5 h, then washed and fixed with 4% paraformaldehyde, and subjected to immunofluorescence with RNF5 antibody. The insets are magnified views of the boxed areas. Scale bar, 20 µm (in cell images) and 10 µm (in the magnified box). (K) Pearson’s coefficient of sRNF5 and sABHD16A-DsRed. n = 30 cells were used for quantification.

Fig 4: ATG2B-RNF5 interaction depends on ATG2B deacetylation.A Acetylation levels of ATG2B were analyzed by western blotting after starvation (ST) treatment. B Acetylation levels of ATG2B were analyzed by western blotting after NAM treatment. C Western blotting analysis of ATG2B protein levels after NAM treatment. D qPCR analysis of ATG2B after NAM treatment. E The influence of NAM on the RNF5 mutual combination of ATG2B was analyzed by western blotting and immunoprecipitation. F Western blotting analysis of ATG2B protein levels after NAM treatment and RNF5 knockdown. G Western blotting analysis of ATG2B protein levels after EX-527 treatment. H The influence of SIRT1 on ATG2B acetylation was analyzed by western blotting and immunoprecipitation. I The influence of SIRT1 on ATG2B protein levels was analyzed by western blotting.