Fig 1: CTSL knockdown promoted complementary activation of CTSB, and CTSB overexpression did not affect autophagic clearance. (A to D) 3T3L1 shLuc (control), as well as shCtsl#1 and shCtsl#2 preadipocytes were differentiated at Day 8 and adipocytes were collected. (A to C) Total cell lysates were analyzed by western blotting using anti-CTSL, LC3, SQSTM1, CIDEC and LMNB1 antibodies (A) with quantitative data shown (B and C). Representative images and the quantitative data (n = 4) were shown. Intensity of LMNB1 was used as a loading control (n = 4). (D) Enzymatic assay of CTSB. Values indicate mean ± SD (n = 4). Differences between values were analyzed by the Student t test. Statistical significance was shown as *P<0.05, **P<0.01. (E to J) 3T3L1 (Mock) or 3T3L1 CTSB (OE) preadipocytes were differentiated into adipocytes at d 8 and treated with 10 μM CA074ME for 24 h. Enzymatic assay of CTSB (E) and CTSL (F) were analyzed by selective substrate. Values indicate mean ± SD. Differences between values were analyzed by Tukey-Kramer method with *P < 0.05, **P < 0.01. (G to J) Total cell lysates analyzed by western blot using anti-FLAG, SQSTM1, LC3, CIDEC, CTSL and LMNB1 antibodies (G) with quantitative data shown (H to J). Representative images and the quantitative data (n = 4) were shown. Intensity of LMNB1 was used as a loading control. Values indicate mean ± SD. Differences between values were analyzed by the Student t test with *P < 0.05, **P < 0.01.
Fig 2: CTSL inhibition impaired autophagic flux in 3T3L1 adipocytes and enhanced CTSB activity. 3T3L1 preadipocytes were differentiated into adipocytes at day 11 and treated with 10 to 100 μM CTSL inhibitor, Z-FY-CHO, or 10 μg/mL cathepsin inhibitor, E64-d. Adipocytes were harvested and analyzed. Enzymatic assay of CTSL (A) and CTSB (B) analyzed by selective substrate. Total cell lysates analyzed by western blot using anti-SQSTM1, CTSL, LC3 and LMNB1 antibodies (C) with quantitative data shown (D and E). Representative images and the quantitative data (n = 4) were shown. Intensity of LMNB1 was used as a loading control (n = 4). 3T3L1 preadipocytes were differentiated into adipocytes at d 11 and treated with 100 μM Z-FY-CHO or 10 μM CTSB inhibitor, CA074ME. Adipocytes were harvested and analyzed. mRNA expression of Ctsl (F) and Ctsb (G) were analyzed by real-time RT-PCR (n = 4). Data were normalized against Rps18 (n = 4). Values indicate mean ± SD. Differences between values were analyzed by Tukey-Kramer method with *P < 0.05, **P < 0.01.
Fig 3: GNS561 induces lysosomal membrane permeabilization and cathepsin-dependent cell death in HepG2 cells. (A) Localization of FITC-dextran after GNS561 treatment for the indicated times. (B) Electron microscopy imaging of lysosomal membrane permeabilization (arrows) after GNS561 treatment (3 µM) for 24 h. (C) Localization of CTSB, CTSD and CTSL after GNS561 treatment for 48 h. (D) Viable cell (ANXA5 (A)-:propidium iodide (PI)-) analysis by flow cytometry of cells pre-treated or not with pepstatin A (Pep A) (5 µM) for 1 h and then treated with Pep A (5 µM) and GNS561 or with GNS561 alone for 48 h. (E) Viable cell (A-:PI-) analysis by flow cytometry of cells pre-treated or not with CA-074-Me (20 µM) for 1 h and then treated with CA-074-Me (20 µM) and GNS561 or with GNS561 alone for 48 h. (F) Viable cell (A-:7-aminoactinomycine D [7AAD]-) analysis by flow cytometry of cells pre-treated or not with Z-Phe (10 µM) for 1 h and then treated with Z-Phe (10 µM) and GNS53-61 or with GNS561 alone for 48 h. Three independent experiments were performed. Data represent the mean + SEM. For comparison, Student t-test was used. For all studies, n = 3 biological replicates. Data represent the mean + SEM. For comparison, Student t-test was used. *represents significant difference, at least p < 0.05.
Fig 4: The lysosomotropic agent GNS561 modulates lysosomal functions in the HepG2 cell line. (A) Chemical labeling of GNS561D in cells. The UV-irradiation activates the aryl azide, and then the click chemistry activates the alkyl azide to create the fluorescent moiety with the dye. (B) Lysosomal localization of GNS561D after NH4Cl pre-treatment (20 mM) for 30 min and then treatment with GNS561D (10 µM) and NH4Cl (20 mM) for 90 min. (C) Cell viability after 24 h of GNS561 exposure in the presence or absence of NH4Cl (20 mM). (D) Staining of lysosomes (LysoTracker) and unbound Zn2+ (Fluozin) after GNS561 treatment (1 h, 10 µM). Quantification of LysoTracker fluorescence in arbitrary units (a.u.) (middle panel) and lysosomal unbound Zn2+ accumulation by Pearson correlation coefficient between LysoTracker and Fluozin (right panel). Fold change of peptidase activity of cysteine cathepsins (including both CTSB-CTSL), CTSB and CTSD after (E) 6 h and (F) 24 h of treatment with GNS561 calculated in comparison with the control condition. (G) Representative immunoblotting of pro-CTSB (precursor form) and mature CTSB (top), pro-CTSL (precursor form) and mature CTSL (middle) and pro-CTSD, intermediate and mature CTSD (bottom) after GNS561 treatment for 16 h. For all blots, GAPDH was used as a loading control. For all studies, n = 3 biological replicates. Data represent the mean + SEM. For comparison, Student t-test was used for (B), (C) and (D), and one-way ANOVA with Dunnett’s post hoc analysis was performed for (E) and (F). *represents significant difference, at least p < 0.05.
Fig 5: Dysregulation of cathepsins was observed in HFD-induced obese WAT. (A) Total protein extracted from WAT of ND, 4HFD, 8HFD and 18HFD mice analyzed by western blot using anti-LAMP2, CTSL, CTSB, SQSTM1, LC3 and GAPDH antibodies (A) with quantitative data shown (B, C, F, G, J and L). Representative images and the quantitative data (n = 4) were shown. Intensity of GAPDH was used as a loading control (n = 4). Enzymatic assay of CTSL (D) and CTSB (H) in WAT of ND, 4HFD, 8HFD and 18HFD mice, as indicated, analyzed by selective substrate. mRNA expression of Ctsl (E), Ctsb (I), Lc3b (K) and Sqstm1 (M) in WAT of ND, 4HFD, 8HFD and 18HFD mice, as indicated, analyzed by real-time RT-PCR (n = 4). Data were normalized against Tbp (n = 4). Values indicate mean ± SD. Differences between values were analyzed by the Student t test with Bonferroni correction *P < 0.05, **P < 0.01.
Supplier Page from Abcam for Anti-Cathepsin L/V/K/H antibody [EPR8011]