Fig 2: SPHK1 induces the EMT by stimulating BECN1 in HepG2 cells. (A, B) Silencing BECN1 blocked cell migration and invasion in SPHK1-overexpressing cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si-BECN1 and plated in the upper chamber of the filters for 24 h. Then cells migrating to the underside of the transwell insert were counted. (C) SPHK1 overexpression regulated the expression of EMT-related markers through stimulating BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si-BECN1 for 24 h and then harvested for cell lysate extraction. The level of epithelial or mesenchymal markers was detected by western blot analysis. (D) Silencing BECN1 blocked the degradation of CDH1 in SPHK1-overexpressing cells. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with pCMV6-CDH1 and si-BECN1 (or control-siRNA) for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were analyzed by western blotting using an anti-CDH1 antibody. (E) Schematic diagram of the mechanism of SPHK1-mediated autophagy and lysosomal CDH1 degradation that stimulates the EMT in HepG2 cells. Con-siRNA, control-siRNA.

Fig 3: Drug combinations reduce the expression of amyloid precursor protein (APP) via autophagy. (A) HCT116 ATG16L1 T300 cells were transfected to express wild type APP or mutant APP proteins, APP715 or APP692. In parallel, cells were transfected with a scrambled siRNA control or with siRNA molecules to knock down the expression of Beclin1 or ATG5. After 24h, cells were treated with vehicle control, neratinib (50 nM), AR12 (2 μM) or the drugs in combination for 6h. Cells were fixed in place and the expression of APPs determined by in-cell immuno-staining. (n = 3 +/-SD). * p < 0.05 less than vehicle control; ** p < 0.05 less than neratinib as a single agent. (B) HCT116 ATG16L1 T300 cells were transfected with plasmids to express wild type APP, APP715 or APP692. After 24h, cells were treated with vehicle control, fingolimod (FTY, 100 nM), MMF (5 μM) or the drugs in combination for 6h or 12h. Cells were fixed in place and the expression of the APPs determined by in-cell immuno-staining. (n = 3 +/-SD). * p < 0.05 less than vehicle control; ** p < 0.05 less than MMF alone value. (C) HCT116 ATG16L1 T300 cells were transfected to express wild type APP or mutant APP proteins, APP715 or APP692. In parallel, cells were transfected with a scrambled siRNA control or with siRNA molecules to knock down the expression of Beclin1 or ATG5. After 24h, cells were treated with vehicle control or with [fingolimod, 100 nM plus MMF, 5 μM]. After 6h cells were fixed in place and the expression of the APPs determined by in-cell immuno-staining. (n = 3 +/-SD). * p < 0.05 less than vehicle control.

Fig 4: Heat stress during the first half of IVM increases the abundance of ATG12 in complex with ATG5. Oocytes collected from aspirated pig follicular fluid underwent in vitro maturation (IVM) in either TN conditions throughout the entire 42-h maturation period (TN/TN), HS conditions during the second half of IVM (TN/HS), or HS conditions during the first half of IVM (HS/TN). Representative western blots for BECN1, ATG12-ATG5 complex, BCL2L1, LC3B, and α-tubulin (A) from oocytes collected before IVM (GV) and after 42 h of IVM. There was increased abundance of ATG12 in complex with ATG5 after 42 h of IVM under the HS/TN temperature treatment compared to the TN/TN and TN/HS (B). Different superscripts within the same protein denotes significant difference (P < 0.05)

Fig 5: Western blotting to detect the expression of autophagy-related proteins. The expression levels of autophagy-related proteins (A and C) Beclin1 and (A and D) LC3-II/I were significantly increased in CLP group compared with the sham group (**P<0.01), while (A and B) the expression level of p62 protein was significantly decreased (*P<0.05). Compared with CLP group, the expression levels of (A and C) Beclin1 and (A and D) LC3-II/I protein were decreased in CLP+3-MA group (*P<0.05, **P<0.01), while (A and B) the expression level of p62 protein was increased (**P<0.01), n=12.