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. 2022 Mar 16;25(4):104085. doi: 10.1016/j.isci.2022.104085

Figure 2.

Figure 2

STX11 affects lipid metabolism through interaction with ATGL

(A) STX11 specifically interacts with ATGL. HeLa cells were co-transfected with STX11-Myc or STX18-Myc and ATGL-HA vectors. STX11-Myc and STX18-Myc proteins were immunoprecipitated using anti-Myc antibodies. STX11 and ATGL proteins in both the immunoprecipitates and lysates were detected by immunoblotting with Myc and HA antibodies.

(B) No co-localization of STX18 and ATGL. HeLa cells were co-transfected with ATGL-HA and STX18-Myc. ATGL-HA protein immunofluorescence staining were performed by using anti-HA (red) and STX18-Myc proteins were stained with anti-Myc (green) antibodies.

(C) Co-localization of STX11 and ATGL in cells. HeLa cells transfected with ATGL-HA and STX11-Myc were incubated under normal growth conditions. Immunofluorescence staining with anti-HA and anti-Myc antibodies was performed to reveal co-localization between ATGL-HA (red) and STX11-Myc (green).

(D and E) Co-localization analysis of STX11 and ATGL using Plot Profile and Co-localization Finder from ImageJ software. Curve coincidence illustrates that the two proteins had co-localized in theory (D) and Pearson’s R = 0.926 (E) (R from 0 to 1.0, 0 indicates no significant correlation and 1.0 indicates complete negative correlation).

(F) STX11 interacts with ATGL in vivo. STX11 proteins were immunoprecipitated using STX11 antibodies in NAFLD mouse liver extracts. STX11 and ATGL proteins were detected by immunoblotting using STX11 and ATGL antibodies.

(G and H) Presence of STX11 inhibits lipolysis mediated by ATGL. STX11-Myc and ATGL-HA co-expression in HeLa cells after treatment with OA (400 μM) for 24 h, and starvation for 4 h. 1: Cell co-expressing STX11 and ATGL; 2: Cell expressing ATGL alone. STX11-Myc and ATGL-HA proteins were subjected to immunostaining with anti-Myc (violet) and anti-HA (red) antibodies. Lipid droplets were stained with BODIPY 493/503 fluorescent dye. Quantification of the number of LDs under the fluorescence microscope (H) (n = 6 cells for Ctrl; STX11 and ATGL co-expression and ATGL overexpression). Data are shown as mean ± SEM (n = 3). ∗∗p < 0.01, t test.