1. Figure S1. Optimization of lysosomal fractionation and TMT intensity of lysosomal proteins. (A) A flow diagram for the preparations of lysosomal isolation and fractions from breast cancer SUM159 cells. (B and C) Isolated fractions analyzed by immunoblotting using antibodies against lysosomal membrane protein LAMP2A, LAMP2, LAMP1, lysosomal matrix protein CTSD, mitochondrial protein TOMM40 and cytosolic protein LDHA. Total protein levels were visualized by Ponceau S Red staining. CMA was induced for 16 h. The lysosomal inhibitor CQ (25 µM) was used to prevent protein degradation in the lysosomes. (D) qPCR data showing the expression of LAMP2A, LAMP2B, LAMP2C and LAMP1 following genetic knockdown using 2 individual LAMP2A siRNAs. (n=3) Data are expressed as mean ± S.D. (E) Fold TMT intensity of lysosomal enrichment of the indicated proteins. (n=4) Data are expressed as mean ± S.D. Adjusted p values. *p<0.05 **p< 0.01 ***p<0.001; Limma package.

2. Figure S2. Translational related proteins EIF4A1, EIF4H and DDX3X are CMA substrates. (A) Expression levels of EIF4A1, EIF4H and DDX3X, following CMA activation in the absence or presence of lysosomal inhibitor (50 µM CQ) in lung NCI-H1792 cancer cells. (B) Time-dependent decrease in protein levels of EIF4A1, EIF4H, DDX3X and, along with 2 known CMA substrates, NFKBIA and HK2, as well as the translation elongation factor EEF2 upon CMA activation up to 36 h in ovarian cancer ES2 cells. (C) The effect of different CMA activators on the expression of EIF4A1, EIF4H and DDX3X in NCI-H1792 lung cancer cells at the indicated time points. Compounds include 6-AN, geldanamycin (HSP90 inhibitor) or AR7, as CMA activators, compared to the combination treatment of AC220 and spautin-1. ACTB was used for equal loading. Immunoblot analysis showing the expression levels of EIF4A1, EIF4H and DDX3X; (D) during glucose-free conditions up to 6 h; (E) upon oxidative stress-induced CMA activation using either H2O2 (250 µM for 4 h) or paraquat (PQ, 2.5 mM for 24 h); (F) following rapamycin or CMA treatment for 24 h, in the indicated cancer cell lines. (G) Expression of WT EIF4A1, EIF4A1Q93,I94A and EIF4A1Q308,K309A proteins with or without CMA activation (AC220 and spautin-1 for 16 h) in ES2 cells. (H) SUnSET western blot showing the incorporation of puromycin-labelling in NCI-H1792 lung cancer cells following treatment with the indicated compounds at the shown time points. Translation inhibitors, CHX and silvestrol, were used as positive controls. Total protein levels visualized by Ponceau S Red staining in the fractions.