Figure 6.

Cathepsin B restricts lysosomal biogenesis and autophagy. (A) Expression analysis of genes encoding cathepsin B, TFEB, WDR45, BECN1, ULK1, and cathepsin E in WT and Ctsb^−/− BMDMs by quantitative real-time PCR. Ctse, cathepsin E; Hprt, hypoxanthine-guanine phosphoribosyltransferase. (B) Immunoblot analysis of TFEB, cathepsin B, and GAPDH (loading control) in uninfected WT and Ctsb^−/− BMDMs. (C) Confocal microscopy analysis of TFEB staining in uninfected WT and Ctsb^−/− BMDMs. (D) Percentage of nuclear localization of TFEB in uninfected WT and Ctsb^−/− BMDMs. At least 200 cells were analyzed for each group. (E) Immunoblot analysis of TFEB in the nuclear and cytoplasmic fractions of uninfected WT and Ctsb^−/− BMDMs (left) and the relative intensity of TFEB in nuclear versus cytoplasmic (right). Lamin B and GAPDH were used as quality controls for nuclear and cytoplasmic fractions separation, respectively. Quantification of the relative protein expression was processed using ImageJ. (F and G) Immunoblot analysis of phosphorylation of mTOR, 4EBP, AMPK, ULK1, and LC3-II in uninfected WT and Ctsb^−/− BMDMs (left) or WT BMDMs in media (Med) with or without 5 µM CA-074 Me (CA) for 2 h. (H) Immunoblot analysis of proteins in F and G in WT BMDMs treated with 5 µM CA-074 Me for the indicated times. (I) Representative images of LysoTracker-labeled WT, Ctsb^−/−, Ctsg^−/−, Elastase^−/−, Nepr3^−/−, and cathepsin B inhibitor treated WT (WT_CA) BMDMs. (J) Immunoblot analysis of AMPK, ULK1, LC3, LAMP1, TFEB, and GAPDH (loading control) in uninfected WT, Ctsg^−/−, Elastase^−/−, and Nepr3^−/− BMDMs. (C and I) Bars, 10 µm. Data are representative of three independent experiments and are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. t-AMPK, total AMPK; t-mTOR, total mTOR.