Fig 2: The regulation of lipophagy by STX11 is mediated through ATGL and SIRT1(A and B) STX11 co-localizes with LC3 under starvation conditions. THLE-2 cells were co-transfected with STX11-mcherry and LC3-EGFP. Cells were treated with starvation (2 h) or no starvation followed by confocal microscopic analysis.Histogram in (B) showing the percentage of STX11 co-localization with LC3 in A (n = 6 cells for control; n = 6 cells for starvation).(C and D) Representative EM images showing LDs (yellow arrows) and autophagosomes (APh) (red arrows) in siSTX11 THLE-2 cells and control THLE-2 cells. Cells were treated with OA (400 µM) 24 h and starved 2 h. Protein expression was analyzed by immunoblotting with anti-STX11 antibodies.(E and F) Quantification of LD and autophagosome (APh) numbers from EM images.(G) Overexpression of STX11 inhibits autophagy. Immunoblotting of THLE-2 cells transfected with STX11-Myc. Cells were treated with OA (400 µM) following serum-free starvation for 2 h. Protein form cell lysates were detected by immunoblotting with anti-STX11, anti-LAMP1, anti-ATGL, and anti-LC3B antibodies.(H) Knockdown of STX11 promotes autophagy. THLE-2 cells were treated with STX11 siRNA. Cell treatments and immunoblotting were the same as described in (G).(I) Presence of STX11 inhibits SIRT1 pathway. Immunoblotting of THLE-2 cells transfected with STX11-Myc. Cell treatments were the same as described in (G). Proteins were detected by immunoblotting with anti-STX11 and anti-SIRT1 antibodies.(J) Deficiency of STX11 activates SIRT1 pathway. THLE-2 cells were treated with STX11 siRNA. Cell treatments and immunoblotting were the same as described in (G).(K) STX11 knockdown activates lipophagy mediated by PKA pathway. THLE-2 cells were treated with STX11 siRNA, and cell treatments were as shown in (G) Immunoblots were assessed with anti-STX11, anti-p-PKA, anti-PKA, anti-SIRT1, anti-ATGL, and anti-LC3B antibodies. ß-actin was used as the loading control for the immunoblots and the data are shown as mean ± SEM (n = 3). NS: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, t test.

Fig 3: The specificity of the involvement of Claudin-5 in autophagy under hypoxia induction. (A) Confocal microscopy images of Claudin-5 (green) and LC3 (purple). Colocalization of Claudin-5 with LC3 (white arrow) and quantitative analysis of LC3 levels by Image-Pro Plus. Mean±SD, n=3 independent experiments per group. Scale bar, 10µm. (B) Western blot analysis of the autophagy markers LC3 and LAMP-1. Mean±SD, n=3 independent experiments per group. *p<0.05 **p<0.01 and ***p<0.005.

Fig 4: Mutant a-synucleinA53T elicits autophagy dysfunction via mTOR-TFEB pathway. (a) Representative immunoblots and quantification of the levels of LAMP1, SQSTM1, and LC3B II in wild-type (WT) or a-synucleinA53T-tg mice. Mean ± SEM, n = 3. (b) Representative immunoblots and quantification of the levels of LAMP1, SQSTM1, and LC3B II in SN47841 cells transfected with Adenovirus-Ctrl/A53T. Mean ± SEM, n = 3. (c) Representative immunoblots and quantification of SQSTM1 and LC3B II levels in SN4741 cells pre-transfected with Adenovirus-A53T and further treated with CQ (10 µM, 4 hr). Means ± SEM, n = 3. (d) Representative images and quantification of Lysosomes (green) in SN4741 cells. Scale bars, 10 µm. (e,f) Levels of TFEB from nuclear fractions, cytoplasmic fractions and total cell lysates in SNpc of mice (e) and SN4741 cell (f) were measured and analyzed. Means ± SEM, n = 3. (g) Representative mTOR staining of SNpc and Cortex tissue of WT and a-synucleinA53T-tg mice. Statistical analysis of the scores of mTOR staining shown in right. Scale bars, 100 µm. (h) Representative immunoblots and quantification of the levels of p-mTOR and mTOR in SN4741 cells. Mean ± SEM, n = 3. (i) SN4741 cells were transfected with Adenovirus-Ctrl/A53T. Then, the interaction between 14-3-3 and TFEB was measured in TFEB Immunoprecipitates. lgG worked as an immunological control. Means ± SEM, n = 3 (the statistical significantly was analyzed by unpaired Student's t test, *p < .05, **p < .01 and ***p < .001). TFEB, transcription factor EB

Fig 5: Lysosomal dysfunction in SH-SY5Y? WDR45 cells. (a) Schematic illustration of dual-fluorescent LC3 assay. The tandem tagged mCherry-GFP-LC3 is a pH-sensitive sensor that is used to monitor the autophagic flux. The GFP tag (green) is acid-sensitive, whereas the mCherry tag (red) is acid-insensitive. In autophagosomes, both GFP and mCherry tags emit fluorescent light resulting in yellow fluorescence. By contrast, the fusion of autophagosomes to lysosomes results in acidic autolysosomes where GFP is quenched, and mCherry continues to emit red fluorescence. (b) Representative confocal images of SH-SY5YWT or SH-SY5Y? WDR45 cells expressing mCherry-GFP-LC3 are shown. SH-SY5YWT cells show mostly red fluorescence in lysosomes because of quenching of GFP at low pH, whereas SH-SY5Y? WDR45 cells display a yellow fluorescence in vesicular structures representing autophagosomes. The blue color indicates nuclei counterstained with DAPI. Scale bar: 10 µm. (c) Representative confocal images of ferritin and LC3 or LAMP1 in SH-SY5Y? WDR45 cells. To detect ferritin, the cells were incubated with an anti-mouse ferritin antibody followed by Alexa Fluor 488-conjugated secondary antibody (green), indicated by arrows. To detect LC3 or LAMP1, the cells were incubated with anti-LC3 antibody or anti-LAMP1 antibody followed by Alexa Fluor 568-conjugated secondary antibody (red). Blue colors indicate nuclei counterstained with DAPI. Scale bar: 10 µm