Supplementary Figure 2: PLIN2 and PLIN3 are CMA substrates.

(a) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells. Two sets of cells are shown. (b) BODIPY 493/503 staining in control cells untreated or treated with ammonium chloride and leupeptin (NL), lactacystin (Lacta) or MG132. Graph: average LD number/cell. n = 5 independent experiments with 40 cells per condition in each experiment. (c) Immunoblot for indicated proteins in total cell lysates from CTR and L2A(−) cells treated or not with OL and lactacystin (Lacta). n = 2 independent experiments. (d,e) CTR and L2A(−) cells expressing DGN or DGN-FS treated or not with OL and Lacta. Graph: average fluorescence intensity/cell. n = 10 fields with 1500 cells per condition from 3 independent experiments. (f,g) Coimmunostaining for PLIN3 and LAMP1 in CTR and L2A(−) cells treated or not with OL, followed by treatment with lysosomal inhibitors (NL). Top: Colocalized pixels in white. Bottom: Merged image of the boxed area at higher magnification. Graph: percentage colocalization of PLIN3 with LAMP1. n = 5 (L2A(−)) and 6 (CTR) independent experiments with 40 cells per condition in each experiment. (h) Immunoblot for indicated proteins of HOM, CMA+ and CMA− lysosomes isolated from fed or starved (Stv) livers of rat untreated or treated with leupeptin (Leup). These blots contributed to the quantification shown in Fig. 2g. All values are mean ± SEM. Differences are significant for ^∗P < 0.05,^∗∗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.