Supplementary Figure 5: Altered cytosolic and lysosomal lipolysis in CMA-incompetent cells.

(a) Coimmunostaining for PLIN2 and ATGL in CTR and L2A(−) cells untreated or treated with OL or incubated with serum-supplemented (OL > S+) or serum-deprived (OL > S−) medium after OL treatment. Insets: Higher magnification areas. Right: Images of the same fields with colocalized pixels shown in white. Graph: percentage colocalization of PLIN2 with ATGL. n = 5 independent experiments with 40 cells per condition in each experiment. (b) Costaining for BODIPY493/503 and ATGL in CTR and L2A(−) cells incubated or not in OL > S+ regular or low glucose media. Bottom: Higher magnification areas. Graph: percentage colocalization of BODIPY with ATGL. n = 5 independent experiments with 40 cells per condition in each experiment. (c) Immunoblot for ATGL of homogenates (HOM) and lipid droplets (LD) isolated from starved wild-type (+) or L2A knockout (−) mice livers (these 3 additional blots support the reproducibility of the blot shown in Fig. 6i). (d) BODIPY493/503 staining in CTR and L2A(−) cells untreated or treated with OL, or treated with 3-methyladenine (3MA) or lysosomal inhibitors ammonium chloride and leupeptin (NL). Higher magnification insets and quantification are shown in Fig. 7a. (e) Immunoblot for LC3 of total cell lysates from CTR and L2A(−) cells treated with OL in the presence or absence of 3-methyladenine (3MA). n = 3 independent experiments. (f) Immunostaining for LC3 in OL-treated CTR and L2A(−) cells. Graph: average number of LC3 puncta/cell. n = 6 independent experiments with 30 cells per condition in each experiment. Values are mean ± SEM. Differences are significant for ^∗∗P < 0.01,^∗∗∗P < 0.001 using Student’s t-test. Uncropped images of blots are shown in Supplementary Fig. 8. Source data is available in Supplementary Table 1.