Journal: The Journal of Clinical Investigation

Article Title: Galectin-3 mediates lysosome-related inflammation within monocyte-derived macrophages in a mouse model of ischemic brain injury

doi: 10.1172/JCI194139

Figure Lengend Snippet: ( A ) Representative immunostaining of cathepsin B/GAL3/CD68 in WT/WT and WT/GAL3 –/– chimera mice 3 d after tMCAO. Scale bar: 50 μm. ( B ) Quantification of cathepsin B + staining areas within CD68 + areas of macrophages. ( C ) Quantification of CD68 + areas. N = 7/group. ( D ) MAP2 staining of brain in vehicle- or CA-074–treated (10 mg/kg, i.v., 2 h after tMCAO and then daily for 2 d) WT/WT or WT/GAL3 –/– chimeric mice 3 d after tMCAO. ( E ) Quantification of brain infarct volume. N = 5–6/group. ( F ) Cathepsin B activity was analyzed using the Magic Red cathepsin kit in GAL3-KO or WT BMDMs with vehicle or TD139 (10 μM) treatment. Scale bar: 50 μm. ( G ) Quantification of the percentages of Magic Red + cells among total cells. N = 6/condition. ( H ) Representative Magic Red staining in WT and GAL3-KO BMDMs treated with or without WT or GAL3-KO lysate for 6 h. Scale bars: 50 μm. ( I ) Quantification of the percentages of Magic Red + cells among total cells. N = 6/condition. Gray blocks show SD. *** P < 0.001 versus (WT BMDM + WT lysate); # P < 0.05, ## P < 0.01 versus (KO BMDM + WT lysate); $ P < 0.05 versus (WT BMDM + KO lysate). ( J ) Immunoblot and quantification of cathepsin B expression in cytosol of BMDMs under the indicated conditions for 6 h. N = 3/condition. ( K ) Coimmunostaining of LAMP2 and cathepsin B in BMDMs under specified conditions for 6 h. Scale bars: 5 μm, 2 μm (zoom). ( L ) Fluorescence intensity profiles of cathepsin B and LAMP2. ( M ) Pearson’s correlation analyses of LAMP2 and cathepsin B. N = 7/condition. ( N ) BMDMs in inserts treated with brain lysates and CA-074 (10 μM) or vehicle were cocultured with OGD neurons. Coverage areas of MAP2-stained neurons were quantified. N = 6/condition. Scale bar: 40 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. Two-tailed, unpaired Student’s t test ( B , C , and E ) or 1-way ( G , J , M , and N ) or 2-way ( I ) ANOVA and Bonferroni’s test.

Article Snippet: Stock solutions of TD139, a specific GAL3 inhibitor (TargetMol) and CA-074 methyl ester, a specific cathepsin B inhibitor (MedChem Express) were prepared at concentrations of 10 mM for in vitro use and 10 mg/mL for in vivo use.

Techniques: Immunostaining, Staining, Activity Assay, Western Blot, Expressing, Fluorescence, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: Galectin-3 mediates lysosome-related inflammation within monocyte-derived macrophages in a mouse model of ischemic brain injury

doi: 10.1172/JCI194139

Figure Lengend Snippet: ( A ) Representative immunostaining of cathepsin B/GAL3/CD68 in WT/WT and WT/GAL3 –/– chimera mice 3 d after tMCAO. Scale bar: 50 μm. ( B ) Quantification of cathepsin B + staining areas within CD68 + areas of macrophages. ( C ) Quantification of CD68 + areas. N = 7/group. ( D ) MAP2 staining of brain in vehicle- or CA-074–treated (10 mg/kg, i.v., 2 h after tMCAO and then daily for 2 d) WT/WT or WT/GAL3 –/– chimeric mice 3 d after tMCAO. ( E ) Quantification of brain infarct volume. N = 5–6/group. ( F ) Cathepsin B activity was analyzed using the Magic Red cathepsin kit in GAL3-KO or WT BMDMs with vehicle or TD139 (10 μM) treatment. Scale bar: 50 μm. ( G ) Quantification of the percentages of Magic Red + cells among total cells. N = 6/condition. ( H ) Representative Magic Red staining in WT and GAL3-KO BMDMs treated with or without WT or GAL3-KO lysate for 6 h. Scale bars: 50 μm. ( I ) Quantification of the percentages of Magic Red + cells among total cells. N = 6/condition. Gray blocks show SD. *** P < 0.001 versus (WT BMDM + WT lysate); # P < 0.05, ## P < 0.01 versus (KO BMDM + WT lysate); $ P < 0.05 versus (WT BMDM + KO lysate). ( J ) Immunoblot and quantification of cathepsin B expression in cytosol of BMDMs under the indicated conditions for 6 h. N = 3/condition. ( K ) Coimmunostaining of LAMP2 and cathepsin B in BMDMs under specified conditions for 6 h. Scale bars: 5 μm, 2 μm (zoom). ( L ) Fluorescence intensity profiles of cathepsin B and LAMP2. ( M ) Pearson’s correlation analyses of LAMP2 and cathepsin B. N = 7/condition. ( N ) BMDMs in inserts treated with brain lysates and CA-074 (10 μM) or vehicle were cocultured with OGD neurons. Coverage areas of MAP2-stained neurons were quantified. N = 6/condition. Scale bar: 40 μm. * P < 0.05, ** P < 0.01, *** P < 0.001. Two-tailed, unpaired Student’s t test ( B , C , and E ) or 1-way ( G , J , M , and N ) or 2-way ( I ) ANOVA and Bonferroni’s test.

Article Snippet: Stock solutions of TD139, a specific GAL3 inhibitor (TargetMol) and CA-074 methyl ester, a specific cathepsin B inhibitor (MedChem Express) were prepared at concentrations of 10 mM for in vitro use and 10 mg/mL for in vivo use.

Techniques: Immunostaining, Staining, Activity Assay, Western Blot, Expressing, Fluorescence, Two Tailed Test

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 4. Effect of pH and protease inhibitors on the endosomal EGF-degrading activity. Soluble endosomal extract (about 1 ng) was incubated with 1026 M EGF at 37 °C for 1 h in 50 mM citrate-phosphate buffer at the indicated pH (A) and for 1 h in 50 mM citrate-phosphate buffer, pH 5, in the absence or presence of 1 mM EDTA, 1 mM 1,10- phenanthroline, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupep- tin, 1027 M E64, 1027 M CA074-Me, or 10 mg/ml pepstatin A (B). At the end of the incubation, the degradation of EGF was measured by HPLC analysis as described in Fig. 2. Results for B are expressed as EGF degraded (percentage of control) after a 1-h incubation and normalized (100%) to that seen in the absence of added compound. Results are the mean 6 SD of three different experiments performed on endosomal fractions prepared from separate liver fractionations.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: Activity Assay, Incubation, Control

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 9. Effect of CA074-Me treatment on the uptake of [125I]EGF in rat liver. Rats were injected with [125I]EGF (107 cpm) having first received an intraperitoneal injection of either 0.15 M NaCl (open bars) or 0.5 mmol CA074-Me (closed bars) 1 h prior to ligand administration. Liver homogenates (A) and endosomal fractions (B) were prepared from rats sacrificed at the indicated times. Recovered radioactivity in the homogenates and endosomal fractions was quanti- fied, and the enrichment of internalized [125I]EGF is expressed by reference to the radioactivity in the homogenates (relative specific activity). The results are means 6 S.D. from three to five fractionations.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: Injection, Radioactivity, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 10. Effect of CA074-Me treat- ment on the internalization of tyro- sine-phosphorylated EGFR following administration of EGF in vivo. Rats were injected with native EGF (5 mg/100 g of body weight), having first received an intraperitoneal injection of either 0.15 M NaCl or 0.5 mmol of CA074-Me 1 h prior to ligand administration. Endosomal frac- tions were isolated at the indicated times and evaluated by Western blotting for their content of EGFR and SHC by using their respective polyclonal IgG and for their immunoreactivity with monoclonal antibody against phosphotyrosine. Each lane contained 20 mg of protein of the endosomal fraction. The arrows on the right indicate the mobility of the EGFR (about 170 kDa) and the three isoforms of SHC (about 66, 55, and 46 kDa). The po- sitions of molecular mass markers are shown at the left.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: In Vivo, Injection, Isolation, Western Blot

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 12. Effect of protease inhibitors on the endosomal EGFR- degrading activity. Rat hepatic endosomal fractions were isolated 15 min after EGF administration (5 mg/100 g of body weight) and incu- bated in isotonic buffer at 37 °C and pH 5 in the absence or presence of 1027 M E64, 1027 M CA074-Me, 10 mg/ml leupeptin, 1 mM EDTA, 0.1 mM (p-hydroxymercuribenzoate), 0.1 mM (p-hydroxymercuriphenylsulfonic acid), 1 mM iodoacetic acid, or 10 mg/ml pepstatin A. After a 2 min incubation, the integrity of the endosome-associated EGFR was evalu- ated by Western blotting. Each lane contains 20 mg of protein of the endosomal fraction. The positions of molecular mass markers are shown at the left.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: Activity Assay, Isolation, Incubation, Western Blot