Fig 2: ?-6 PUFAs activate autophagy and antioxidation via TOR-dependent and TOR-independent signaling pathways.The levels of total and phosphorylated raptor, TSC2, ULK1, ATG13, and TFEB and the levels of nuclear Nrf2 were examined by western blot analysis and quantitated after treatment with a TOR pathway inhibitor (500 nM RAPA) or activator (2 µM MHY1485) (n = 3). 17 gels were run and 17 blots were made. All of the blots were not stripped and re-probed. The blots of T-raptor, P-raptorSer792, T-TSC2, P-TSC2Ser1387, T-ULK1, P-ULK1Ser467, P-ULK1Ser555, P-ULK1Ser637, P-ULK1Ser757, T-ATG13, P-ATG13Ser355, T-TFEB, P-TFEBSer211, and T-Nrf2 were used for the GAPDH loading controls and the blots of n-Nrf2 was used for the LaminB loading controls (a, b). Representative confocal microscopic image of hepatocytes stained with LysoTracker after treatment with a TOR pathway inhibitor or activator. Scale bars, 50 µm. The same type of samples (LA group) was used in TOR pathway inhibitor (RAPA) group and autophagy inhibitor (3-MA) group (c, e). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, and the numbers of the autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity after treatment with a TOR pathway inhibitor or activator (d, f). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed after treatment with a TOR pathway inhibitor or activator (n = 6) (g, h). The results are presented as the mean with SEM and were analyzed using Tukey’s test. Bars bearing the same letters are not significantly different among treatments (*P = 0.05).

Fig 3: High glucose (HG) induced lysosomal Ca2+-dependent lysosomal exocytosis and interleukin-1ß (IL-1ß) secretion via calcineurin/transcription factor EB (TFEB) pathway in U937 cells. (A) The percentage of ß-hexosaminidase activity was induced by glycyl-l-phenylalanine-b-napthylamide (GPN). U937 cells were treated with GPN (400 µM) in the presence of GAPDH small interfering RNA (siRNA) or TFEB siRNA or BAPTA (10 µM) (n = 4). The results were normalized to control. (B) Representative immunoblots for TFEB and ß-actin in GAPDH siRNA (GAPDH si)- or TFEB siRNA-treated cells (TFEB si) (n = 3). (C) The relative median fluorescence intensity (MFI) of surface lysosomal-associated membrane protein-1 (LAMP-1) staining that were in the presence of GAPDH or TFEB siRNA under low glucose (LG; 5.5 mM glucose) or HG (30 mM glucose for 48 h) (n = 4). (D) The relative gene expressions of LAMP-1, cathepsin D, and cathepsin B in the presence of GAPDH or TFEB siRNA under LG or HG (30 mM glucose for 48 h) in U937 cells (n = 5). (E) ELISA for IL-1ß secretion from the supernatants of U937 cells that were pre-treated with FK506 (25 µM) or cyclosporin A (Cs A; 10 µM), or in the presence of GAPDH siRNA (GA si) or TFEB siRNA (TFEB si) under LG or HG. (F) The relative gene expressions of IL-1ß in the presence of GAPDH or TFEB siRNA under HG in U937 cells (n = 4). (G,H) Representative immunoblots for pro-IL-1ß, IL-1ß p17, or pro-caspase-1, cleaved caspase-1 (p20) and ß-actin in the presence of GAPDH or TFEB siRNA under LG or HG (30 mM glucose for 48 h) in U937 cells (n = 4). Data were shown as mean ± SEM. (A) *P < 0.05 and **P < 0.01 vs. GPN + GAPDH siRNA; ##P < 0.01 vs. GPN. (C,E) **P < 0.01 vs. LG; #P < 0.05 and ##P < 0.01 vs. HG. (D,F) **P < 0.01 and ***P < 0.001 vs. LG + GAPDH siRNA; ##P < 0.01 and ###P < 0.001 vs. HG + GAPDH siRNA.

Fig 4: VP plus MG co-treatment targets P53 to lysosomes. Immunoblots showing LC3B-II (a), TFEB (b), P62 (c.i) and P53 (c.ii.) expressions after VP (10 µM) or VP plus MG treatment for 24 h. The ‘third bracket’ represents an HMW band detected. d and e Immunoblots showing LAMP-2A and HSC70 expression after VP or VP plus MG treatment. (f.i) Immunofluorescence image showing P53 (FITC-green) and Lysotracker-Red; (f.ii) or P53 (FITC-green) and LAMP-2A (TRITC-red) post VP and MG treatment. White arrow indicates co-localization. Scale bar: 10 μm. Objective: Plan Apochromat 63x/1.40 oil M27

Fig 5: miR-21 inhibits VMP1 expression through PTEN/AKT/TFEB pathway.a The protein expression level of PTEN, phosphorylated AKT (Ser473), total AKT, and VMP1 were measured by Western blot. b RKO-miR-21-KO was transfected with miR-21 nucleotide mimic, and the mRNA expression level of miR-21 and VMP1 was measured by qPCR, the protein expression level of PTEN, phosphorylated AKT (Ser473), total AKT, and VMP1 were measured by Western blot. c RKO and RKO-miR-21-KO were transfected with siRNA targeting PTEN respectively, the protein expression level of PTEN, phosphorylated AKT (Ser473), total AKT, and VMP1 were measured by Western blot. d RKO and RKO-miR-21-KO were treated with 2 µM VO-Ohpic and 1 µM Afuresertib for 24 h, the protein expression level of phosphorylated AKT (Ser473), total AKT, and VMP1 in after were measured by Western blot. e RKO and RKO-miR-21-KO cells with no treat or treat with 2 µM VO-Ohpic for 24 h, the protein level of TFEB in cytoplasm and nucleus respectively was measured by Western blot, H3 and β-actin were used as nuclear and cytosolic markers, respectively. f RKO and RKO-miR-21-KO cells with no treat or treat with 1 µM Afuresertib for 24 h, the protein level of TFEB in cytoplasm and nucleus respectively was measured by Western blot, H3 and Tubulin were used as nuclear and cytosolic markers, respectively. g The protein expression level of TFEB and VMP1 were measured by Western blot after overexpression of the constitutively active TFEB by pCIP-caTfeb vector or knock-down of TFEB expression by siRNA in RKO cells. Data are presented as mean ± SD. ***P < 0.001.