Fig 2: K45R-AMBRA1 mutant maintains AMBRA1 stability and promotes PIK3C3 activity. (A) Scheme for lysines on AMBRA1. (B, C) K45 is the RNF2-mediated ubiquitination site on AMBRA1. Various AMBRA1 truncations (B) or mutants (C) were co-transfected with RNF2 with or without HA-tagged ubiquitin (HA-Ub) into MEFs for 24 h. Cells were harvested after pretreatment with 10 µM MG132 for 6 h and EBSS for 2 h, followed by IP with antibody against AMBRA1. The immunoprecipitates were dissociated with 1% SDS and reimmunoprecipitated with anti-FLAG. Immunoprecipitates were immunoblotted with the indicated antibodies. (D) K45R-AMBRA1 enhances autophagy. AMBRA1-silenced MEFs were stably rescued with WT-AMBRA1 or K45R-AMBRA1 and starved in EBSS for the indicated times, followed by IP with antibody against FLAG after pretreated with 10 µM MG132 for 6 h. Immunoprecipitates were immunoblotted with the indicated antibodies. (E) The half-life of AMBRA1. AMBRA1-silenced MEFs were transfected with WT-AMBRA1 (upper panel) or K45R-AMBRA1 (lower panel) and selected for stable expression with 2 µg/ml puromycin. Cells were then treated with or without EBSS for the indicated times in the presence of 20 µg/ml CHX before harvest for immunoblotting with the indicated antibodies. (F) The association of BECN1 with PIK3C3 is enhanced in K45R-AMBRA1-rescued cells. AMBRA1-silenced MEFs were rescued with WT-AMBRA1 or K45R-AMBRA1 for 24 h, followed by IP with anti-BECN1 antibody. Immunoprecipitates were immunoblotted with the indicated antibodies (upper panel). The ratios of PIK3C3 versus BECN1 were calculated and shown in the lower panel. (G) K45R-AMBRA1 augments PIK3C3 kinase activity. AMBRA1-silenced MEFs were rescued with WT-AMBRA1 or K45R-AMBRA1, followed by starvation in EBSS for the indicated times. Cells were lysed for immunoblotting with anti-ATG14 antibody for autophagy-specific PIK3C3. Immunoprecipitates were split into two equal parts, one for a loading control and the other for in vitro kinase assay. Radioactive PI(3)P was resolved by thin layer chromatography (TLC), and normalized against the BECN1-associated PIK3C3. (H, I) K45R-AMBRA1 promotes autophagic activity. AMBRA1-silenced MEFs were rescued with WT-AMBRA1 or K45R-AMBRA1, followed by starvation in EBSS for 2 h with or without 20 nM BafA1. Cells were lysed for immunoblotting with the indicated antibodies (H). Endogenous MAP1LC3B were stained with antibody against MAP1LC3B and MAP1LC3B dots were counted (I). (J) RNF2 deficiency does not affect the K45R-AMBRA1-elevated autophagy. AMBRA1-silenced MEFs were stably rescued with WT-AMBRA1 or K45R-AMBRA1, followed by starvation in EBSS for 2 h with or without 20 nM BafA1. Cells were lysed for immunoblotting with the indicated antibodies. Data are representative of at least three separate experiments and shown as means ± SD. Scale bar, 10 µm. **P< 0.01; ***P< 0.001.

Fig 3: Effects of resveratrol on mitophagy induction following OGD/R in primary cortical neurons. (A) Representative immunoblots and data for LC3 and mitochondrial markers, including TOMM20 and TIMM23, as determined by western blotting. GAPDH was used as the loading control. (B) Representative immunostaining of cells for LC3B (green) and mitochondria with anti-TOM20 (red). Scale bar, 10 µm. Data are presented as the mean ± SEM (n=3). *P<0.05 vs. the control. #P<0.05 vs. OGD/R. OGD/R, oxygen/glucose deprivation/reoxygenation; TOMM, translocase of outer mitochondrial membrane; TIMM, translocase of inner mitochondrial membrane.

Fig 4: MBH FKBP51 affects sympathetic outflow and peripheral autophagy signaling.FKBP51 overexpression is depicted in blue, and FKBP51 deletion is depicted in green. (A and B) Representative decrease in tissue NE content after a-MPT injection (left) and turnover rate (right) were determined on SM and eWAT (see fig. S8 for pancreas, heart, iWAT, and BAT tissues). Quantification of (C) pAMPK (T172) and (D) pp70S6K (T389), and (E) p62 level in the SM and eWAT. (F) Representative blots. (G to H) FKBP51 overexpression increased autophagic flux and in SM and eWAT. (I) Representative blots of chloroquine the experiment. Quantification of (J) pAMPK (T172), (K) pp70S6K (T389), (L) LC3B-II, and (M) p62 levels in SM and eWAT in animals lacking FKBP51 in the MBH. (N) Representative blots of FKBP51MBH-KO protein analysis. All data are shown as ±SEM. Protein data are shown as the relative protein expression compared to control. A two-way ANOVA was performed, followed by a Tukey’s multiple comparison test in (F) and (G). For (A) to (E) and (I) to (L), an unpaired Student’s t test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Lactobacillus rhamnosus restores the impaired autophagic flux induced by F. nucleatum in vitro. (A) Representative LC3B and SQSTM1/p62 western blots of Caco2 cells treated with F. nucleatum. supernatant at increasing MOIs for 12 h. GAPDH was used as a loading control. (B) Caco2 cells challenged with F. nucleatum were pre-treated with or without CQ. Western blot analysis was conducted to detect the LC3B level. (C) Protein levels of LC3B and SQSTM1/p62 of Caco2 cells treated with F. nucleatum. and Lactobacillus rhamnosus supernatant for 12 h. (D) Histogram of relative expression of LC3B and p62. (E) Caco2 cells challenged with Lactobacillus rhamnosus were pre-treated with or without CQ. Western blot analysis was conducted to detect the LC3B level. *P=0.05, **P=0.01. F. nucleatum/F.n., Fusobacterium nucleatum; L.r., Lactobacillus rhamnosus; CQ, chloroquine.