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: STX11 directly interacts with ATGL(A) Schematic representation of the structure of murine ATGL, its mutants, and the PT domain in ATGL is indicated as the gray region.(B) STX11 interacts with the PT domain of ATGL. HEK293T cells were co-transfected with STX11-HA together with different ATGL constructs (ATGL-Myc + STX11-HA, ATGL-PT-Myc + STX11-HA, ATGL-?PT-Myc + STX11-HA). STX11-HA proteins were immunoprecipitated with anti-HA antibodies. STX11 and ATGL proteins in immunoprecipitated lysates were detected by immunoblotting with HA and Myc antibodies.(C) Schematic representation of the structure of murine STX11, its mutants, and the Habc domain and the SNARE motif in STX11 are indicated as the gray region.(D) C-terminal of STX11 interacts with the PT domain of ATGL. HEK293T cells were co-transfected with STX11?C-HA and ATGL-PT-Myc. Other groups and immunoprecipitation and immunoblotting analyses were performed as described in (B).(E) STX11 co-localizes with PT domain of ATGL. Immunofluorescence staining of STX11-HA and ATGL-PT-Myc in HeLa cells was performed using anti-HA (green) and anti-Myc (red) antibodies. Intensity traces from the original images were plotted using ImageJ software. Curve coincidence illustrates that the two proteins have co-localized in theory.(F) HeLa cells were co-transfected with STX11?C-HA and ATGL-Myc. STX11?C-HA proteins immunofluorescence staining was performed by using anti-HA (green) and ATGL-Myc proteins were stained with anti-Myc (red) antibodies.(G and H) GST pulldown analysis between recombinant STX11-GST, ATGL-MBP-His, and ATGL-MBP-PT-His.

Fig 4: STX11 affects the spatial localization of ATGL(A and B) Live-cell confocal imaging of HeLa cells transiently expressing STX11-EGFP and ATGL-mcherry cultured under basal growth conditions or treated with isoproterenol (1 µM) (see corresponding Video S1). STX11 and ATGL co-localization analysis using ImageJ software (B) (n = 3 cells for live-cell confocal imaging).(C and D) Representative images and quantification of ATGL co-localization with LDs. HeLa cells were transfected with ATGL-HA and incubated with OA (400 µM) 24 h, followed by treatment with 1 µM isoproterenol for 30 min or starvation for 4 h. ATGL were co-stained with anti-HA (red) and Lipid droplets were co-stained with BODIPY 493/503. Histogram in (D) showing the percentage of ATGL co-localization with LDs in C (n = 18 cells for isoproterenol; n = 21 cells for starvation).(E and F) Representative images and quantification of ATGL co-localization with LDs demonstrate that STX11 prevents ATGL movement to the surface of LDs. STX11-Myc and ATGL-HA co-expressed in HeLa cells and treated as in (C). STX11-Myc protein immunofluorescence staining was performed using anti-Myc (violet) antibodies and ATGL-HA proteins were stained with anti-HA (red) antibodies. Lipid droplets were stained with BODIPY 493/503 fluorescent dye. Histogram in (F) showing the percentage of ATGL co-localization with LDs in E (n = 18 cells for isoproterenol; n = 22 cells for starvation). Data are shown as mean ± SEM (n = 3). ***p < 0.001, t test.

Fig 5: Co-localization of STX11 with ATGL at the ER(A) STX11 localizes to the ER. THLE-2 cells were transfected with STX11-mcherry, and the ER was stained with ER-Tracker Blue-White DPX followed by confocal microscopic analysis.(B) No significant co-localization of ATGL with the ER. THLE-2 cells were transfected with ATGL-mcherry and then the ER were stained with ER-Tracker Blue-White DPX followed by confocal microscopic analysis.(C) Analysis of STX11 and ATGL co-localization with ER is shown as Rr.(D) Analysis using confocal microscopy demonstrates that co-localization of STX11 with ATGL at the ER. STX11-EGFP and ATGL-mcherry were co-transfected into THLE-2 cells and ER were stained with ER-Tracker Blue-White DPX.(E) Schematic of the amino acid mutation in the C-terminal region of STX11.(F) No co-localization of STX11-C5A and ATGL. HEK293T cells were co-transfected with ATGL-HA together with STX11-Myc or STX11-C5A-Myc and were immunoprecipitated with anti-Myc antibodies. STX11 and ATGL proteins in immunoprecipitated lysates were detected by immunoblotting with Myc and HA antibodies.(G) HeLa cells were co-transfected with STX11-C5A-Myc and ATGL-HA. STX11-C5A-Myc protein immunofluorescence staining was performed using anti-Myc (green) and ATGL-HA proteins were stained with anti-HA (red) antibodies.(H) Co-localization of STX11 and DGAT2 in cells (white arrows). HeLa cells were co-transfected with STX11-Myc and DGAT2-HA. Immunofluorescence staining with anti-HA and anti-Myc antibodies was performed to reveal co-localization between DGAT2-HA (green) and STX11-Myc (red).(I) STX11 and DGAT2 have a direct physical interaction. STX11-Myc proteins were immunoprecipitated with anti-Myc antibodies. STX11 and DGAT2 proteins in immunoprecipitates and lysates were detected by immunoblotting using Myc and HA antibodies. Data are shown as mean ± SEM (n = 3).