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  • 86
    Thermo Fisher gene exp ctsb hs00157194 m1
    ASMase activity increases concordantly with <t>CtsB</t> and CtsD expression during HSC activation. A: Time-course of ASMase and NSMase activity during mouse HSC activation in culture. B: Time-course of α-SMA, CtsB, and CtsD expression in primary mouse
    Gene Exp Ctsb Hs00157194 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ctsb
    Lysosomal alterations in SAD fibroblasts. ( A ) Quantification of the amount of lysosomes per cell. ( B ) Quantification of the lysosomal acidity represented by the ratio between untreated and bafilomycin (100 nM)-treated cells. The following healthy/AD age-matched samples were used: AG1136/AG05809, AG07803/AG06262 and AG05813/AG06263. Every measurement was performed in triplicate, and the graphs represent the means and standard deviations of the average of every cell line. ( C ) Representative western blot of <t>LAMP1</t> protein in the absence or presence of CCCP (20 μM) and quantification of the levels under basal conditions. The following couples were used: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. ( D ) Representative western blot analysis of the protein levels of Cathepsin <t>B/CTSB</t> and quantification of the protein levels of CTSB under basal conditions. The following four couples were used in this assay: AG11362/AG05809, AG07803/AG06262, AG05813/AG06263 and AG07310/AG06869. ( E ). The activity was normalized by the maximum fluorescence detected in the last time point of the healthy sample. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. Every measurement was performed in triplicate, and the average obtained for each cell line was used for the graph. ( F ) Representative western blot analysis of SQSTM1 and quantification of the levels in the presence of NH 4 Cl with respect to the absence. The following couples were used: AG11362/AG05809, AG07310/AG06869 and AG07803/AG06262. ( G ) Representative confocal microscopy images of healthy and AD fibroblasts expressing mRFP-EGFP-LC3. ( H ) Quantification of autophagolysosomes per cell corresponding to the percentage of only red structures. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. All graphs show means and standard deviations of the mentioned healthy/AD age-matched couple samples. † P
    Ctsb, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems ctsb
    Lysosomal alterations in SAD fibroblasts. ( A ) Quantification of the amount of lysosomes per cell. ( B ) Quantification of the lysosomal acidity represented by the ratio between untreated and bafilomycin (100 nM)-treated cells. The following healthy/AD age-matched samples were used: AG1136/AG05809, AG07803/AG06262 and AG05813/AG06263. Every measurement was performed in triplicate, and the graphs represent the means and standard deviations of the average of every cell line. ( C ) Representative western blot of <t>LAMP1</t> protein in the absence or presence of CCCP (20 μM) and quantification of the levels under basal conditions. The following couples were used: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. ( D ) Representative western blot analysis of the protein levels of Cathepsin <t>B/CTSB</t> and quantification of the protein levels of CTSB under basal conditions. The following four couples were used in this assay: AG11362/AG05809, AG07803/AG06262, AG05813/AG06263 and AG07310/AG06869. ( E ). The activity was normalized by the maximum fluorescence detected in the last time point of the healthy sample. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. Every measurement was performed in triplicate, and the average obtained for each cell line was used for the graph. ( F ) Representative western blot analysis of SQSTM1 and quantification of the levels in the presence of NH 4 Cl with respect to the absence. The following couples were used: AG11362/AG05809, AG07310/AG06869 and AG07803/AG06262. ( G ) Representative confocal microscopy images of healthy and AD fibroblasts expressing mRFP-EGFP-LC3. ( H ) Quantification of autophagolysosomes per cell corresponding to the percentage of only red structures. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. All graphs show means and standard deviations of the mentioned healthy/AD age-matched couple samples. † P
    Ctsb, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co ctsb inhibitor
    In vitro functional analyses of CST6 overexpression. (A) CST6 mRNA levels and CM protein levels in SCP2 control and overexpression cells. (B) Overexpressed CST6 inhibited the enzymatic activities of <t>CTSB</t> and <t>CTSL.</t> The cathepsin inhibitors epoxysuccinyl peptide (CA074) and Z-FY(t-Bu)-DMK (FY) were used as controls. (C-G) In vitro growth rates (C) , soft-agar colony formation (D) , wound-healing (E) , transwell migration (F) and invasion (G) of SCP2 control and overexpression cells. (H) CST6 overexpression in MCF10CA1h and the transwell invasion analysis. Student's t -test * P
    Ctsb Inhibitor, supplied by Merck & Co, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Shanghai Genechem sirnas against ctsb
    In vitro functional analyses of CST6 overexpression. (A) CST6 mRNA levels and CM protein levels in SCP2 control and overexpression cells. (B) Overexpressed CST6 inhibited the enzymatic activities of <t>CTSB</t> and <t>CTSL.</t> The cathepsin inhibitors epoxysuccinyl peptide (CA074) and Z-FY(t-Bu)-DMK (FY) were used as controls. (C-G) In vitro growth rates (C) , soft-agar colony formation (D) , wound-healing (E) , transwell migration (F) and invasion (G) of SCP2 control and overexpression cells. (H) CST6 overexpression in MCF10CA1h and the transwell invasion analysis. Student's t -test * P
    Sirnas Against Ctsb, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti ctsb
    In vitro functional analyses of CST6 overexpression. (A) CST6 mRNA levels and CM protein levels in SCP2 control and overexpression cells. (B) Overexpressed CST6 inhibited the enzymatic activities of <t>CTSB</t> and <t>CTSL.</t> The cathepsin inhibitors epoxysuccinyl peptide (CA074) and Z-FY(t-Bu)-DMK (FY) were used as controls. (C-G) In vitro growth rates (C) , soft-agar colony formation (D) , wound-healing (E) , transwell migration (F) and invasion (G) of SCP2 control and overexpression cells. (H) CST6 overexpression in MCF10CA1h and the transwell invasion analysis. Student's t -test * P
    Anti Ctsb, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc c9 tagged ctsb
    In vitro functional analyses of CST6 overexpression. (A) CST6 mRNA levels and CM protein levels in SCP2 control and overexpression cells. (B) Overexpressed CST6 inhibited the enzymatic activities of <t>CTSB</t> and <t>CTSL.</t> The cathepsin inhibitors epoxysuccinyl peptide (CA074) and Z-FY(t-Bu)-DMK (FY) were used as controls. (C-G) In vitro growth rates (C) , soft-agar colony formation (D) , wound-healing (E) , transwell migration (F) and invasion (G) of SCP2 control and overexpression cells. (H) CST6 overexpression in MCF10CA1h and the transwell invasion analysis. Student's t -test * P
    C9 Tagged Ctsb, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Abcam anti ctsb
    Increased lysosomal hydrolytic capacity induced by chronic or acute AEP-deficiency does not depend on TFEB activation. a Cellular localisation of TFEB-GFP in HKC-8 cells treated or untreated with 50 μM MVO26630 or 1 μM Torin1 overnight. (Scale bar = 20 μm). Data are the average ± SD of n = 93 cells for control sample, n = 48 for Torin1-treated sample and n = 92 for MVO-treated sample. Statistical significance was calculated using a Dunnett’s multiple comparison test. b Localisation of TFEB by IF in WT and AEP −/− kidney sections. (Scale bar = 20 μm). c Levels of P-4EBP1 by IF in WT and AEP −/− kidney sections. (Scale bar = 20 μm). d Immunoblotting and quantitation of LC3II/total <t>LC3</t> ratio in lysates from WT MEFs treated with Rapamycin (1 μM) or Torin1 (1 μM) for 4 h or with MVO26630 (50 μM) for 16 h. Data are the average ± SD of n = 5 biologically independent samples. Statistical significance was calculated using a Dunnett’s multiple comparison test. e Levels of expression of <t>CtsB</t> and CtsD in HKC-8 WT and TFEB knockout Exon 1 treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 6 biologically independent samples for CtsB and n = 7 biologically independent samples for CtsD. Statistical significance was calculated using a two-sided unpaired t- test. f mRNA expression levels for CtsB and CtsD in HKC-8 WT and TFEB knockout Exon 1 treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 3 biologically independent samples. Full data and gel calibration markers can be seen in Supplementary Fig. 3c . Statistical significance was calculated using a two-sided unpaired t- test
    Anti Ctsb, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Addgene inc ctsb vector
    CTSL knockdown promoted complementary activation of <t>CTSB,</t> and CTSB <t>overexpression</t> did not affect autophagic clearance. (A to D) 3T3L1 sh Luc (control), as well as sh Ctsl #1 and sh Ctsl #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
    Ctsb Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher ctsb sirna
    Cathepsin B knockdown in OC2 and CAL27 cells reduce cell migration. Western blot analysis showing the cathepsin B Knockdown efficiency in (A) OC2 cell and (B) CAL27 oral cancer cell lines. Detection of cell migration ability by transfected with control <t>siRNA</t> or <t>CTSB</t> siRNA in (C) OC2 cell and (D) CAL27 cell. Results shown that cathepsin B siRNA significantly reduced the migration of OC2 and CAL27 cells. *p
    Ctsb Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Enzo Biochem anti ctsb
    Cathepsin B knockdown in OC2 and CAL27 cells reduce cell migration. Western blot analysis showing the cathepsin B Knockdown efficiency in (A) OC2 cell and (B) CAL27 oral cancer cell lines. Detection of cell migration ability by transfected with control <t>siRNA</t> or <t>CTSB</t> siRNA in (C) OC2 cell and (D) CAL27 cell. Results shown that cathepsin B siRNA significantly reduced the migration of OC2 and CAL27 cells. *p
    Anti Ctsb, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore ctsb inhibitor
    IFNγ release after iNKT activation in vitro . (A,B) antigen-presenting cells (APCs) (LX2 and C1R-CD1d) were preincubated with or without Cathepsin B <t>(CTSB)</t> and Cathepsin S (CTSS) inhibitors (75 and 7.5 µM, respectively, for 1 h) before being treated with <t>α-GalCer</t> (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the αGC-charged APCs (C1R-CD1d or LX2 cells) with human primary iNKT cells for 24 h, and IFNγ was determined in the extracellular media by enzyme-linked immunosorbent assay (ELISA) (C,D) , APCs (LX2 and C1R-CD1d) were treated with α-GalCer (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the α-GalCer-charged APCs with human primary iNKT cells in the presence or absence of CTSB or CTSS inhibitors for 24 h, and IFNγ was determined in the extracellular media by ELISA. Results are given as a mean ± SD of five independent experiments; * p
    Ctsb Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti ctsb antibody
    IFNγ release after iNKT activation in vitro . (A,B) antigen-presenting cells (APCs) (LX2 and C1R-CD1d) were preincubated with or without Cathepsin B <t>(CTSB)</t> and Cathepsin S (CTSS) inhibitors (75 and 7.5 µM, respectively, for 1 h) before being treated with <t>α-GalCer</t> (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the αGC-charged APCs (C1R-CD1d or LX2 cells) with human primary iNKT cells for 24 h, and IFNγ was determined in the extracellular media by enzyme-linked immunosorbent assay (ELISA) (C,D) , APCs (LX2 and C1R-CD1d) were treated with α-GalCer (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the α-GalCer-charged APCs with human primary iNKT cells in the presence or absence of CTSB or CTSS inhibitors for 24 h, and IFNγ was determined in the extracellular media by ELISA. Results are given as a mean ± SD of five independent experiments; * p
    Anti Ctsb Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp ctsb hs00947433 m1
    Changes in protease gene expression caused by EcTI treatment The EcTI effects on the protease expression in U87 cells were measured by qRT-PCR analysis and compared to non-treated control (c) cells.The effect of EcTI on the expression of the metalloproteases Mmp2 (A) , Mmp9 (B) , Mmp14 (C) , urokinase plasminogen activator, Plau (D) and its receptor, PlauR (E) , Calpain 1, Capn1 (F) , Calpain 2, Capn2 (G) , Cathepsin L, CtsL (H) , and Cathepsin B, <t>CtsB</t> (I) . Error bars indicate SD. * p
    Gene Exp Ctsb Hs00947433 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher gene exp ctsb rn00575030 m1
    Changes in protease gene expression caused by EcTI treatment The EcTI effects on the protease expression in U87 cells were measured by qRT-PCR analysis and compared to non-treated control (c) cells.The effect of EcTI on the expression of the metalloproteases Mmp2 (A) , Mmp9 (B) , Mmp14 (C) , urokinase plasminogen activator, Plau (D) and its receptor, PlauR (E) , Calpain 1, Capn1 (F) , Calpain 2, Capn2 (G) , Cathepsin L, CtsL (H) , and Cathepsin B, <t>CtsB</t> (I) . Error bars indicate SD. * p
    Gene Exp Ctsb Rn00575030 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Merck & Co ctsb
    Changes in protease gene expression caused by EcTI treatment The EcTI effects on the protease expression in U87 cells were measured by qRT-PCR analysis and compared to non-treated control (c) cells.The effect of EcTI on the expression of the metalloproteases Mmp2 (A) , Mmp9 (B) , Mmp14 (C) , urokinase plasminogen activator, Plau (D) and its receptor, PlauR (E) , Calpain 1, Capn1 (F) , Calpain 2, Capn2 (G) , Cathepsin L, CtsL (H) , and Cathepsin B, <t>CtsB</t> (I) . Error bars indicate SD. * p
    Ctsb, supplied by Merck & Co, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Human Protein Atlas ctsb
    Changes in protease gene expression caused by EcTI treatment The EcTI effects on the protease expression in U87 cells were measured by qRT-PCR analysis and compared to non-treated control (c) cells.The effect of EcTI on the expression of the metalloproteases Mmp2 (A) , Mmp9 (B) , Mmp14 (C) , urokinase plasminogen activator, Plau (D) and its receptor, PlauR (E) , Calpain 1, Capn1 (F) , Calpain 2, Capn2 (G) , Cathepsin L, CtsL (H) , and Cathepsin B, <t>CtsB</t> (I) . Error bars indicate SD. * p
    Ctsb, supplied by Human Protein Atlas, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad ctsb
    Changes in protease gene expression caused by EcTI treatment The EcTI effects on the protease expression in U87 cells were measured by qRT-PCR analysis and compared to non-treated control (c) cells.The effect of EcTI on the expression of the metalloproteases Mmp2 (A) , Mmp9 (B) , Mmp14 (C) , urokinase plasminogen activator, Plau (D) and its receptor, PlauR (E) , Calpain 1, Capn1 (F) , Calpain 2, Capn2 (G) , Cathepsin L, CtsL (H) , and Cathepsin B, <t>CtsB</t> (I) . Error bars indicate SD. * p
    Ctsb, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Neuromics ctsb
    Sorting, transport and localization of lysosomal proteins in cultured PT ki hepatocytes. A , Isolated hepatocytes from wt and PT ki mice were cultivated for 2 days and the expression of Neu1, Npc2, Ctsl, <t>Ctsb,</t> Ctsd, <t>Ctsz</t> and Lamp1 were determined by Western blotting in the cell lysates and conditioned media. The ER marker protein disulfide isomerase (Pdi) was used as loading control. All soluble lysosomal proteins are missorted to variable degree indicated by reduced intracellular levels and increased secretion into the medium. The intracellular retention of Ctsl, Ctsb and Ctsd are less affected. Comparable Lamp1 levels between wt and PT ki cells are observed. B , Hepatocytes from wt and PT ki mice were labeled with [ 35 S]-methioine for 45 min and either harvested (−, lanes 1, 3 ) or chased for 5 h (+, lanes 2, 4 ) followed by immunoprecipitation of cathepsin D (Ctsd) and cathepsin Z (Ctsz) from cell extracts ( lanes 1–4 ) and media ( lanes 5–6 ). The immunocomplexes were solubilized, separated by SDS-PAGE and visualized by fluorography. Secretion of newly synthesized Ctsd and Ctsz is increased in PT ki hepatocytes, accompanied by a significant reduction or complete loss of processed intracellular mature forms, respectively, during the chase. C , Isolated hepatocytes from wt and PT ki mice were fixed, and immunostained for localization of endogenous cathepsin D ( Ctsd ), cathepsin B ( Ctsb ) and neuraminidase 1 ( Neu1 ) in red and the lysosomal marker protein Lamp1 ( green ). Nuclei were stained with DAPI ( blue ). Only merged images are shown in which yellow indicates colocalization of the antigens. Magnified images of the indicated white rectangles are shown in the insets. Scale bars : 7.5 μm.
    Ctsb, supplied by Neuromics, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher gene exp ctsb mm01310506 m1
    Predominantly cytosolic TFEB and abnormal levels of phosphorylated TFE 3 in RPE cells from fasted and fed Cryba1 KO mice. (A) Western blot showing increased levels of TFEB in Cryba1 fl/fl nuclear extracts following fasting. TFEB is predominantly localized in the cytosol (C) in cells from normally fed Cryba1 fl/fl (control) mice, but after fasting the nuclear (N) proportion increases. In cells from Cryba1 KO mice, significantly lower nuclear levels of TFEB were observed in both fasted and fed conditions. (B) Cryba1 fl/fl and Cryba1 KO cells were subjected to qPCR analysis using Taqman probes for some CLEAR (coordinated lysosomal expression and regulation) genes after fasting. Cryba1 KO cells showed a reduction in the transcript levels for lysosomal hydrolases: CTSD (55%), <t>CTSB</t> (75%); lysosomal acidification: ATP 6V0A1 (56%); lysosomal membrane proteins: LAMP 1 (15%), LAMP 2 (40%), and MCOLIN 1 (35%); and gene related to autophagy: BECN 1 (51%), UVRAG (35%). The graph shows mean ± SD from triplicate experiments, representative of at least three independent experiments. (two‐tailed t‐test) (C) Western blot for p‐ TFE 3 in RPE isolated from Cryba1 fl/fl and Cryba1 KO mice. The data indicate decreased p‐ TFE 3 in controls, but increased p‐ TFE 3 in KO RPE after fasting. (n = 4) Quantification of C is shown in D. (E) Western blot showing cathepsin D (both precursor and immature forms) expression in the RPE from four‐month‐old (n = 6) and 10‐month‐old (n = 3) Cryba1 KO mice vs. floxed controls. F and G show densitometric quantification for data in E. (H) CTSD immunostaining (red) and DAPI staining (blue) on RPE flat mounts from Cryba1 fl/fl and Cryba1 KO mice at 2 and 10 months of age (n = 3). There are fewer fluorescent puncta in Cryba1 KO RPE cells, than in Cryba1 fl/fl RPE at both ages. Scale bars=10 μm. (I) TEM of RPE in 20‐month‐old Cryba1 fl/fl mouse showing photoreceptor outer segments ( POS ) and RPE (left panel). Unlike Cryba1 KO mouse, TEM (center panel) showed numerous vacuoles with possible accumulation of lipid‐like droplets (arrow). TEM also showed many autolysosomes, some with incomplete degradation (arrow) retained in 20‐month‐old Cryba1 KO mouse (right panel). (J) Western blot and quantification (K) of protein level of SQSTM 1 (p62) in RPE of 10‐month‐old Cryba1 KO mice relative to control mice. (n = 3) * P
    Gene Exp Ctsb Mm01310506 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher gene exp ctsb mm01310507 g1
    Predominantly cytosolic TFEB and abnormal levels of phosphorylated TFE 3 in RPE cells from fasted and fed Cryba1 KO mice. (A) Western blot showing increased levels of TFEB in Cryba1 fl/fl nuclear extracts following fasting. TFEB is predominantly localized in the cytosol (C) in cells from normally fed Cryba1 fl/fl (control) mice, but after fasting the nuclear (N) proportion increases. In cells from Cryba1 KO mice, significantly lower nuclear levels of TFEB were observed in both fasted and fed conditions. (B) Cryba1 fl/fl and Cryba1 KO cells were subjected to qPCR analysis using Taqman probes for some CLEAR (coordinated lysosomal expression and regulation) genes after fasting. Cryba1 KO cells showed a reduction in the transcript levels for lysosomal hydrolases: CTSD (55%), <t>CTSB</t> (75%); lysosomal acidification: ATP 6V0A1 (56%); lysosomal membrane proteins: LAMP 1 (15%), LAMP 2 (40%), and MCOLIN 1 (35%); and gene related to autophagy: BECN 1 (51%), UVRAG (35%). The graph shows mean ± SD from triplicate experiments, representative of at least three independent experiments. (two‐tailed t‐test) (C) Western blot for p‐ TFE 3 in RPE isolated from Cryba1 fl/fl and Cryba1 KO mice. The data indicate decreased p‐ TFE 3 in controls, but increased p‐ TFE 3 in KO RPE after fasting. (n = 4) Quantification of C is shown in D. (E) Western blot showing cathepsin D (both precursor and immature forms) expression in the RPE from four‐month‐old (n = 6) and 10‐month‐old (n = 3) Cryba1 KO mice vs. floxed controls. F and G show densitometric quantification for data in E. (H) CTSD immunostaining (red) and DAPI staining (blue) on RPE flat mounts from Cryba1 fl/fl and Cryba1 KO mice at 2 and 10 months of age (n = 3). There are fewer fluorescent puncta in Cryba1 KO RPE cells, than in Cryba1 fl/fl RPE at both ages. Scale bars=10 μm. (I) TEM of RPE in 20‐month‐old Cryba1 fl/fl mouse showing photoreceptor outer segments ( POS ) and RPE (left panel). Unlike Cryba1 KO mouse, TEM (center panel) showed numerous vacuoles with possible accumulation of lipid‐like droplets (arrow). TEM also showed many autolysosomes, some with incomplete degradation (arrow) retained in 20‐month‐old Cryba1 KO mouse (right panel). (J) Western blot and quantification (K) of protein level of SQSTM 1 (p62) in RPE of 10‐month‐old Cryba1 KO mice relative to control mice. (n = 3) * P
    Gene Exp Ctsb Mm01310507 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ctsb  (Bachem)
    89
    Bachem ctsb
    Predominantly cytosolic TFEB and abnormal levels of phosphorylated TFE 3 in RPE cells from fasted and fed Cryba1 KO mice. (A) Western blot showing increased levels of TFEB in Cryba1 fl/fl nuclear extracts following fasting. TFEB is predominantly localized in the cytosol (C) in cells from normally fed Cryba1 fl/fl (control) mice, but after fasting the nuclear (N) proportion increases. In cells from Cryba1 KO mice, significantly lower nuclear levels of TFEB were observed in both fasted and fed conditions. (B) Cryba1 fl/fl and Cryba1 KO cells were subjected to qPCR analysis using Taqman probes for some CLEAR (coordinated lysosomal expression and regulation) genes after fasting. Cryba1 KO cells showed a reduction in the transcript levels for lysosomal hydrolases: CTSD (55%), <t>CTSB</t> (75%); lysosomal acidification: ATP 6V0A1 (56%); lysosomal membrane proteins: LAMP 1 (15%), LAMP 2 (40%), and MCOLIN 1 (35%); and gene related to autophagy: BECN 1 (51%), UVRAG (35%). The graph shows mean ± SD from triplicate experiments, representative of at least three independent experiments. (two‐tailed t‐test) (C) Western blot for p‐ TFE 3 in RPE isolated from Cryba1 fl/fl and Cryba1 KO mice. The data indicate decreased p‐ TFE 3 in controls, but increased p‐ TFE 3 in KO RPE after fasting. (n = 4) Quantification of C is shown in D. (E) Western blot showing cathepsin D (both precursor and immature forms) expression in the RPE from four‐month‐old (n = 6) and 10‐month‐old (n = 3) Cryba1 KO mice vs. floxed controls. F and G show densitometric quantification for data in E. (H) CTSD immunostaining (red) and DAPI staining (blue) on RPE flat mounts from Cryba1 fl/fl and Cryba1 KO mice at 2 and 10 months of age (n = 3). There are fewer fluorescent puncta in Cryba1 KO RPE cells, than in Cryba1 fl/fl RPE at both ages. Scale bars=10 μm. (I) TEM of RPE in 20‐month‐old Cryba1 fl/fl mouse showing photoreceptor outer segments ( POS ) and RPE (left panel). Unlike Cryba1 KO mouse, TEM (center panel) showed numerous vacuoles with possible accumulation of lipid‐like droplets (arrow). TEM also showed many autolysosomes, some with incomplete degradation (arrow) retained in 20‐month‐old Cryba1 KO mouse (right panel). (J) Western blot and quantification (K) of protein level of SQSTM 1 (p62) in RPE of 10‐month‐old Cryba1 KO mice relative to control mice. (n = 3) * P
    Ctsb, supplied by Bachem, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher gene exp ctsb mm01310508 g1
    Predominantly cytosolic TFEB and abnormal levels of phosphorylated TFE 3 in RPE cells from fasted and fed Cryba1 KO mice. (A) Western blot showing increased levels of TFEB in Cryba1 fl/fl nuclear extracts following fasting. TFEB is predominantly localized in the cytosol (C) in cells from normally fed Cryba1 fl/fl (control) mice, but after fasting the nuclear (N) proportion increases. In cells from Cryba1 KO mice, significantly lower nuclear levels of TFEB were observed in both fasted and fed conditions. (B) Cryba1 fl/fl and Cryba1 KO cells were subjected to qPCR analysis using Taqman probes for some CLEAR (coordinated lysosomal expression and regulation) genes after fasting. Cryba1 KO cells showed a reduction in the transcript levels for lysosomal hydrolases: CTSD (55%), <t>CTSB</t> (75%); lysosomal acidification: ATP 6V0A1 (56%); lysosomal membrane proteins: LAMP 1 (15%), LAMP 2 (40%), and MCOLIN 1 (35%); and gene related to autophagy: BECN 1 (51%), UVRAG (35%). The graph shows mean ± SD from triplicate experiments, representative of at least three independent experiments. (two‐tailed t‐test) (C) Western blot for p‐ TFE 3 in RPE isolated from Cryba1 fl/fl and Cryba1 KO mice. The data indicate decreased p‐ TFE 3 in controls, but increased p‐ TFE 3 in KO RPE after fasting. (n = 4) Quantification of C is shown in D. (E) Western blot showing cathepsin D (both precursor and immature forms) expression in the RPE from four‐month‐old (n = 6) and 10‐month‐old (n = 3) Cryba1 KO mice vs. floxed controls. F and G show densitometric quantification for data in E. (H) CTSD immunostaining (red) and DAPI staining (blue) on RPE flat mounts from Cryba1 fl/fl and Cryba1 KO mice at 2 and 10 months of age (n = 3). There are fewer fluorescent puncta in Cryba1 KO RPE cells, than in Cryba1 fl/fl RPE at both ages. Scale bars=10 μm. (I) TEM of RPE in 20‐month‐old Cryba1 fl/fl mouse showing photoreceptor outer segments ( POS ) and RPE (left panel). Unlike Cryba1 KO mouse, TEM (center panel) showed numerous vacuoles with possible accumulation of lipid‐like droplets (arrow). TEM also showed many autolysosomes, some with incomplete degradation (arrow) retained in 20‐month‐old Cryba1 KO mouse (right panel). (J) Western blot and quantification (K) of protein level of SQSTM 1 (p62) in RPE of 10‐month‐old Cryba1 KO mice relative to control mice. (n = 3) * P
    Gene Exp Ctsb Mm01310508 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ASMase activity increases concordantly with CtsB and CtsD expression during HSC activation. A: Time-course of ASMase and NSMase activity during mouse HSC activation in culture. B: Time-course of α-SMA, CtsB, and CtsD expression in primary mouse

    Journal: The American Journal of Pathology

    Article Title: Acidic Sphingomyelinase Controls Hepatic Stellate Cell Activation and in Vivo Liver Fibrogenesis

    doi: 10.2353/ajpath.2010.091257

    Figure Lengend Snippet: ASMase activity increases concordantly with CtsB and CtsD expression during HSC activation. A: Time-course of ASMase and NSMase activity during mouse HSC activation in culture. B: Time-course of α-SMA, CtsB, and CtsD expression in primary mouse

    Article Snippet: Predesigned TaqMan assays for target genes from Applied Biosystems were used (CtsB, Hs00157194_m1; ASMase, Hs00609415_m1; and 18S, Hs99999901_s1).

    Techniques: Activity Assay, Expressing, Activation Assay

    Increased Expression of ASMase and CtsB in Livers from Patients with NASH

    Journal: The American Journal of Pathology

    Article Title: Acidic Sphingomyelinase Controls Hepatic Stellate Cell Activation and in Vivo Liver Fibrogenesis

    doi: 10.2353/ajpath.2010.091257

    Figure Lengend Snippet: Increased Expression of ASMase and CtsB in Livers from Patients with NASH

    Article Snippet: Predesigned TaqMan assays for target genes from Applied Biosystems were used (CtsB, Hs00157194_m1; ASMase, Hs00609415_m1; and 18S, Hs99999901_s1).

    Techniques: Expressing

    ASMase inhibition decreases CtsB, CtsD, and α-SMA expression as well as proliferation in the human LX2 cell line. A: ASMase activity after inhibition with imipramine (Imip, 25 μmol/L) or amitriptyline (Ami, 25 μmol/L) for 24 hours

    Journal: The American Journal of Pathology

    Article Title: Acidic Sphingomyelinase Controls Hepatic Stellate Cell Activation and in Vivo Liver Fibrogenesis

    doi: 10.2353/ajpath.2010.091257

    Figure Lengend Snippet: ASMase inhibition decreases CtsB, CtsD, and α-SMA expression as well as proliferation in the human LX2 cell line. A: ASMase activity after inhibition with imipramine (Imip, 25 μmol/L) or amitriptyline (Ami, 25 μmol/L) for 24 hours

    Article Snippet: Predesigned TaqMan assays for target genes from Applied Biosystems were used (CtsB, Hs00157194_m1; ASMase, Hs00609415_m1; and 18S, Hs99999901_s1).

    Techniques: Inhibition, Expressing, Activity Assay

    Heterozygous ASMase +/− HSCs exhibit decreased expression of CtsB, CtsD, and α-SMA, and reduced proliferation in culture. A: ASMase activity in cultured 7-day-old wild-type or heterozygous ASMase +/− HSCs. B: Time

    Journal: The American Journal of Pathology

    Article Title: Acidic Sphingomyelinase Controls Hepatic Stellate Cell Activation and in Vivo Liver Fibrogenesis

    doi: 10.2353/ajpath.2010.091257

    Figure Lengend Snippet: Heterozygous ASMase +/− HSCs exhibit decreased expression of CtsB, CtsD, and α-SMA, and reduced proliferation in culture. A: ASMase activity in cultured 7-day-old wild-type or heterozygous ASMase +/− HSCs. B: Time

    Article Snippet: Predesigned TaqMan assays for target genes from Applied Biosystems were used (CtsB, Hs00157194_m1; ASMase, Hs00609415_m1; and 18S, Hs99999901_s1).

    Techniques: Expressing, Activity Assay, Cell Culture

    ASMase inhibition or siRNA transfection reduces HSC activation and proliferation. A: ASMase activity after inhibition with imipramine (Imip, 25 μmol/L) for 24 hours or ASMase siRNA silencing for 48 hours, in cultured 7-days old mouse HSCs. CtsB

    Journal: The American Journal of Pathology

    Article Title: Acidic Sphingomyelinase Controls Hepatic Stellate Cell Activation and in Vivo Liver Fibrogenesis

    doi: 10.2353/ajpath.2010.091257

    Figure Lengend Snippet: ASMase inhibition or siRNA transfection reduces HSC activation and proliferation. A: ASMase activity after inhibition with imipramine (Imip, 25 μmol/L) for 24 hours or ASMase siRNA silencing for 48 hours, in cultured 7-days old mouse HSCs. CtsB

    Article Snippet: Predesigned TaqMan assays for target genes from Applied Biosystems were used (CtsB, Hs00157194_m1; ASMase, Hs00609415_m1; and 18S, Hs99999901_s1).

    Techniques: Inhibition, Transfection, Activation Assay, Activity Assay, Cell Culture

    ASMase and CtsB expression are increased in patients with NASH. Liver samples from control or NASH subjects were processed for RNA isolation to determine the expression of ASMase mRNA ( A ) and CtsB mRNA ( B ). Patients with NASH were separated in two groups:

    Journal: The American Journal of Pathology

    Article Title: Acidic Sphingomyelinase Controls Hepatic Stellate Cell Activation and in Vivo Liver Fibrogenesis

    doi: 10.2353/ajpath.2010.091257

    Figure Lengend Snippet: ASMase and CtsB expression are increased in patients with NASH. Liver samples from control or NASH subjects were processed for RNA isolation to determine the expression of ASMase mRNA ( A ) and CtsB mRNA ( B ). Patients with NASH were separated in two groups:

    Article Snippet: Predesigned TaqMan assays for target genes from Applied Biosystems were used (CtsB, Hs00157194_m1; ASMase, Hs00609415_m1; and 18S, Hs99999901_s1).

    Techniques: Expressing, Isolation

    Lysosomal alterations in SAD fibroblasts. ( A ) Quantification of the amount of lysosomes per cell. ( B ) Quantification of the lysosomal acidity represented by the ratio between untreated and bafilomycin (100 nM)-treated cells. The following healthy/AD age-matched samples were used: AG1136/AG05809, AG07803/AG06262 and AG05813/AG06263. Every measurement was performed in triplicate, and the graphs represent the means and standard deviations of the average of every cell line. ( C ) Representative western blot of LAMP1 protein in the absence or presence of CCCP (20 μM) and quantification of the levels under basal conditions. The following couples were used: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. ( D ) Representative western blot analysis of the protein levels of Cathepsin B/CTSB and quantification of the protein levels of CTSB under basal conditions. The following four couples were used in this assay: AG11362/AG05809, AG07803/AG06262, AG05813/AG06263 and AG07310/AG06869. ( E ). The activity was normalized by the maximum fluorescence detected in the last time point of the healthy sample. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. Every measurement was performed in triplicate, and the average obtained for each cell line was used for the graph. ( F ) Representative western blot analysis of SQSTM1 and quantification of the levels in the presence of NH 4 Cl with respect to the absence. The following couples were used: AG11362/AG05809, AG07310/AG06869 and AG07803/AG06262. ( G ) Representative confocal microscopy images of healthy and AD fibroblasts expressing mRFP-EGFP-LC3. ( H ) Quantification of autophagolysosomes per cell corresponding to the percentage of only red structures. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. All graphs show means and standard deviations of the mentioned healthy/AD age-matched couple samples. † P

    Journal: Human Molecular Genetics

    Article Title: PARK2 enhancement is able to compensate mitophagy alterations found in sporadic Alzheimer's disease

    doi: 10.1093/hmg/ddv616

    Figure Lengend Snippet: Lysosomal alterations in SAD fibroblasts. ( A ) Quantification of the amount of lysosomes per cell. ( B ) Quantification of the lysosomal acidity represented by the ratio between untreated and bafilomycin (100 nM)-treated cells. The following healthy/AD age-matched samples were used: AG1136/AG05809, AG07803/AG06262 and AG05813/AG06263. Every measurement was performed in triplicate, and the graphs represent the means and standard deviations of the average of every cell line. ( C ) Representative western blot of LAMP1 protein in the absence or presence of CCCP (20 μM) and quantification of the levels under basal conditions. The following couples were used: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. ( D ) Representative western blot analysis of the protein levels of Cathepsin B/CTSB and quantification of the protein levels of CTSB under basal conditions. The following four couples were used in this assay: AG11362/AG05809, AG07803/AG06262, AG05813/AG06263 and AG07310/AG06869. ( E ). The activity was normalized by the maximum fluorescence detected in the last time point of the healthy sample. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. Every measurement was performed in triplicate, and the average obtained for each cell line was used for the graph. ( F ) Representative western blot analysis of SQSTM1 and quantification of the levels in the presence of NH 4 Cl with respect to the absence. The following couples were used: AG11362/AG05809, AG07310/AG06869 and AG07803/AG06262. ( G ) Representative confocal microscopy images of healthy and AD fibroblasts expressing mRFP-EGFP-LC3. ( H ) Quantification of autophagolysosomes per cell corresponding to the percentage of only red structures. The couples used for this experiment were: AG11362/AG05809, AG05813/AG06263 and AG07310/AG06869. All graphs show means and standard deviations of the mentioned healthy/AD age-matched couple samples. † P

    Article Snippet: The primary antibodies used were: TOMM20 (sc-11415, Santa Cruz Biotechnology, Santa Cruz, C A, USA), COX4I1 (complex IV) (459600, Invitrogen, Eugene, OR, USA), LC3 (B7931, Sigma, St. Louis, MO, USA, for WB), LC3 (M152-3, MBL, Woburn, MA, USA, for immunostaining), SQSTM1 (610832, BD Biosciences, San Jose, CA, USA), BECN1 (sc-11427, Santa Cruz), Ubiquitin (Z0458, Dako, Glostrup, Denmark), LAMP1 (611042, BD Biosciences), CTSB (AB4064, Chemicon, Darmstadt, Germany), GAPDH (ab8245, Abcam, Cambridge, UK), PARK2 (sc-32282, Santa Cruz), PINK1 (BC100-494, Novus, Cambridge, UK) and SCaMC-1 [short calcium-binding mitochondrial carrier 1, kindly given by Dr Araceli del Arco ( )].

    Techniques: Western Blot, Activity Assay, Fluorescence, Confocal Microscopy, Expressing

    In vitro functional analyses of CST6 overexpression. (A) CST6 mRNA levels and CM protein levels in SCP2 control and overexpression cells. (B) Overexpressed CST6 inhibited the enzymatic activities of CTSB and CTSL. The cathepsin inhibitors epoxysuccinyl peptide (CA074) and Z-FY(t-Bu)-DMK (FY) were used as controls. (C-G) In vitro growth rates (C) , soft-agar colony formation (D) , wound-healing (E) , transwell migration (F) and invasion (G) of SCP2 control and overexpression cells. (H) CST6 overexpression in MCF10CA1h and the transwell invasion analysis. Student's t -test * P

    Journal: Cell Research

    Article Title: Differential secretome analysis reveals CST6 as a suppressor of breast cancer bone metastasis

    doi: 10.1038/cr.2012.90

    Figure Lengend Snippet: In vitro functional analyses of CST6 overexpression. (A) CST6 mRNA levels and CM protein levels in SCP2 control and overexpression cells. (B) Overexpressed CST6 inhibited the enzymatic activities of CTSB and CTSL. The cathepsin inhibitors epoxysuccinyl peptide (CA074) and Z-FY(t-Bu)-DMK (FY) were used as controls. (C-G) In vitro growth rates (C) , soft-agar colony formation (D) , wound-healing (E) , transwell migration (F) and invasion (G) of SCP2 control and overexpression cells. (H) CST6 overexpression in MCF10CA1h and the transwell invasion analysis. Student's t -test * P

    Article Snippet: The CTSB inhibitor (CA-074, Merck) and the CTSL inhibitor (Z-FY[t-Bu]-DMK, Merck) were used as controls.

    Techniques: In Vitro, Functional Assay, Over Expression, Migration

    Increased lysosomal hydrolytic capacity induced by chronic or acute AEP-deficiency does not depend on TFEB activation. a Cellular localisation of TFEB-GFP in HKC-8 cells treated or untreated with 50 μM MVO26630 or 1 μM Torin1 overnight. (Scale bar = 20 μm). Data are the average ± SD of n = 93 cells for control sample, n = 48 for Torin1-treated sample and n = 92 for MVO-treated sample. Statistical significance was calculated using a Dunnett’s multiple comparison test. b Localisation of TFEB by IF in WT and AEP −/− kidney sections. (Scale bar = 20 μm). c Levels of P-4EBP1 by IF in WT and AEP −/− kidney sections. (Scale bar = 20 μm). d Immunoblotting and quantitation of LC3II/total LC3 ratio in lysates from WT MEFs treated with Rapamycin (1 μM) or Torin1 (1 μM) for 4 h or with MVO26630 (50 μM) for 16 h. Data are the average ± SD of n = 5 biologically independent samples. Statistical significance was calculated using a Dunnett’s multiple comparison test. e Levels of expression of CtsB and CtsD in HKC-8 WT and TFEB knockout Exon 1 treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 6 biologically independent samples for CtsB and n = 7 biologically independent samples for CtsD. Statistical significance was calculated using a two-sided unpaired t- test. f mRNA expression levels for CtsB and CtsD in HKC-8 WT and TFEB knockout Exon 1 treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 3 biologically independent samples. Full data and gel calibration markers can be seen in Supplementary Fig. 3c . Statistical significance was calculated using a two-sided unpaired t- test

    Journal: Nature Communications

    Article Title: Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis

    doi: 10.1038/s41467-018-07741-6

    Figure Lengend Snippet: Increased lysosomal hydrolytic capacity induced by chronic or acute AEP-deficiency does not depend on TFEB activation. a Cellular localisation of TFEB-GFP in HKC-8 cells treated or untreated with 50 μM MVO26630 or 1 μM Torin1 overnight. (Scale bar = 20 μm). Data are the average ± SD of n = 93 cells for control sample, n = 48 for Torin1-treated sample and n = 92 for MVO-treated sample. Statistical significance was calculated using a Dunnett’s multiple comparison test. b Localisation of TFEB by IF in WT and AEP −/− kidney sections. (Scale bar = 20 μm). c Levels of P-4EBP1 by IF in WT and AEP −/− kidney sections. (Scale bar = 20 μm). d Immunoblotting and quantitation of LC3II/total LC3 ratio in lysates from WT MEFs treated with Rapamycin (1 μM) or Torin1 (1 μM) for 4 h or with MVO26630 (50 μM) for 16 h. Data are the average ± SD of n = 5 biologically independent samples. Statistical significance was calculated using a Dunnett’s multiple comparison test. e Levels of expression of CtsB and CtsD in HKC-8 WT and TFEB knockout Exon 1 treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 6 biologically independent samples for CtsB and n = 7 biologically independent samples for CtsD. Statistical significance was calculated using a two-sided unpaired t- test. f mRNA expression levels for CtsB and CtsD in HKC-8 WT and TFEB knockout Exon 1 treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 3 biologically independent samples. Full data and gel calibration markers can be seen in Supplementary Fig. 3c . Statistical significance was calculated using a two-sided unpaired t- test

    Article Snippet: HKC-8 WT AND TFEB KOs Starvation Total cell extracts from HKC-8 WT and TFEB KO cells starved or not for 4 h in HBSS at 37 °C and 10% CO2 were prepared in complete RIPA buffer and equal amounts of protein were separated by SDS-PAGE and subjected to direct Western blotting using anti-LC3 or anti-CtsB as described above.

    Techniques: Activation Assay, Quantitation Assay, Expressing, Knock-Out

    Chronic or acute inhibition of cysteine or aspartyl cathepsins lead to a similar response. a Levels of P-STAT3 and CtsD in WT MEFs treated or not with 10 μM E64 or 5 μM Pepstatin A for 72 h. Data are the average ± SD of n = 4 biologically independent samples for P-STAT3 and n = 3 biologically independent samples for CtsD. Statistical significance was calculated using a two-sided unpaired t- test. b Levels of P-STAT3, STAT3, CtsD and SOD1 in CtsB −/− heart or CtsL −/− heart compared to WT heart. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test

    Journal: Nature Communications

    Article Title: Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis

    doi: 10.1038/s41467-018-07741-6

    Figure Lengend Snippet: Chronic or acute inhibition of cysteine or aspartyl cathepsins lead to a similar response. a Levels of P-STAT3 and CtsD in WT MEFs treated or not with 10 μM E64 or 5 μM Pepstatin A for 72 h. Data are the average ± SD of n = 4 biologically independent samples for P-STAT3 and n = 3 biologically independent samples for CtsD. Statistical significance was calculated using a two-sided unpaired t- test. b Levels of P-STAT3, STAT3, CtsD and SOD1 in CtsB −/− heart or CtsL −/− heart compared to WT heart. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test

    Article Snippet: Antibodies against Laminin (ab11575), CtsD (ab75852), CtsB (ab92955), Lamp1 (ab24170) and Fibronectin (ab199056) were purchased from AbCam.

    Techniques: Inhibition

    Increased lysosomal hydrolytic capacity induced by chronic or acute AEP-deficiency is controlled by the Jak2-STAT3 signalling pathway. a STAT3 activation in WT but not AEP −/− MEFs treated for 16 h with increasing concentrations of MVO26630. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a Dunnett’s multiple comparison test. b P-STAT3 localisation in WT and AEP −/− MEFs treated or not with 50 μM MVO26630 for 16 h (endogenous P-STAT3) and HKC-8 cells transfected with STAT3-YFP and treated or not with 50 μM MVO26630 for 16 h. Data are the average ± SEM of n = 96 cells for control samples and n = 86 for MVO-treated samples. Statistical significance was calculated using a two-sided unpaired t- test. (Scale bar = 20μm). c STAT3 activation in HKC-8 cells treated with increasing levels of MVO26630 overnight. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. d CtsD, CtsB and P-STAT3 levels in WT MEFs compared to WT MEFs treated overnight with 50 μM MVO26630 with or without 100 μM of the STAT3 inhibitor S3I-201. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. e Levels of STAT3, CtsB and CtsD in WT HKC-8 cells transfected with a non-targeting siRNA (NT) or two different STAT3-targeting siRNAs (#8 and #10) and treated or not with 50 μM MVO26630 for 16 h. Data are the average ± SD of n ≥ 3. Statistical significance was calculated using a two-sided unpaired t- test. f P-STAT3 western blot in WT HKC-8 treated with 50 μM MVO26630 in the presence or absence of the Jak2 inhibitor SD-1029 (10 μM). Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a Dunnett’s multiple comparison test. g Levels of P-Jak2 in WT HKC-8 cells treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. h Levels of Jak2, P-STAT3, CtsB and CtsD in WT HKC-8 cells transfected with a non-targeting siRNA (NT) or two different Jak2-targeting siRNAs (#6 and #7) and treated or not with 50 μM MVO26630 for 16 h. Data are the average ± SD of n ≥ 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test

    Journal: Nature Communications

    Article Title: Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis

    doi: 10.1038/s41467-018-07741-6

    Figure Lengend Snippet: Increased lysosomal hydrolytic capacity induced by chronic or acute AEP-deficiency is controlled by the Jak2-STAT3 signalling pathway. a STAT3 activation in WT but not AEP −/− MEFs treated for 16 h with increasing concentrations of MVO26630. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a Dunnett’s multiple comparison test. b P-STAT3 localisation in WT and AEP −/− MEFs treated or not with 50 μM MVO26630 for 16 h (endogenous P-STAT3) and HKC-8 cells transfected with STAT3-YFP and treated or not with 50 μM MVO26630 for 16 h. Data are the average ± SEM of n = 96 cells for control samples and n = 86 for MVO-treated samples. Statistical significance was calculated using a two-sided unpaired t- test. (Scale bar = 20μm). c STAT3 activation in HKC-8 cells treated with increasing levels of MVO26630 overnight. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. d CtsD, CtsB and P-STAT3 levels in WT MEFs compared to WT MEFs treated overnight with 50 μM MVO26630 with or without 100 μM of the STAT3 inhibitor S3I-201. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. e Levels of STAT3, CtsB and CtsD in WT HKC-8 cells transfected with a non-targeting siRNA (NT) or two different STAT3-targeting siRNAs (#8 and #10) and treated or not with 50 μM MVO26630 for 16 h. Data are the average ± SD of n ≥ 3. Statistical significance was calculated using a two-sided unpaired t- test. f P-STAT3 western blot in WT HKC-8 treated with 50 μM MVO26630 in the presence or absence of the Jak2 inhibitor SD-1029 (10 μM). Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a Dunnett’s multiple comparison test. g Levels of P-Jak2 in WT HKC-8 cells treated or untreated with 50 μM MVO26630 for 16 h. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. h Levels of Jak2, P-STAT3, CtsB and CtsD in WT HKC-8 cells transfected with a non-targeting siRNA (NT) or two different Jak2-targeting siRNAs (#6 and #7) and treated or not with 50 μM MVO26630 for 16 h. Data are the average ± SD of n ≥ 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test

    Article Snippet: Antibodies against Laminin (ab11575), CtsD (ab75852), CtsB (ab92955), Lamp1 (ab24170) and Fibronectin (ab199056) were purchased from AbCam.

    Techniques: Activation Assay, Transfection, Western Blot

    Direct STAT3 involvement in regulation of lysosomal hydrolase genes under conditions of protease activity/lysosomal substrate imbalance. a Agarose gel analysis of representative ChIP-PCR products for AEP, CtsD and CtsL promoters in WT and AEP-deficient MEFs. Full data and gel calibration markers can be seen in Supplementary Fig. 6 . b Quantitative PCR analysis of DNA from STAT3 ChIP of WT (blue circles) and AEP-deficient (red squares) MEFs expressed as fold enrichment. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. c AEP activity in WT MEF treated overnight with different concentrations of MVO26630 in the presence (dashed line) or absence (solid line) of 100 μM S3I-201. Data are the average ± SD of three independent experiments. d Quantitative PCR analysis of the mRNA levels for different lysosomal proteases in WT (blue circles) and STAT3c 3T3 (red squares) cells. Data are the average ± SD of n = 6 biologically independent samples for all samples, except for AEP where n = 5 and CtsL where n = 3. Statistical significance was calculated using a two-sided unpaired t- test. e Quantitative PCR analysis of the mRNA levels for different lysosomal proteases in WT MEFs treated (red squares) or not (blue circles) with 30 mg/ml BSA overnight. Data are the average ± SD of n = 4 biologically independent samples for all samples, except for AEP and CtsD where n = 3. Statistical significance was calculated using a two-sided unpaired t- test. f AEP and CtsD/E activities measured in WT and STAT3c 3T3 cells. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t -test. g Activities of AEP, CtsB/L and CtsD/E in WT MEF treated or not with 30 mg/ml BSA overnight in the presence or absence of 100 μM S3I-201. Data are the average ± SD of n = 3 biologically independent samples. h Quantitation of P-STAT3 and LC3II levels in WT MEFs treated with Rapamycin (1 μM) or Torin1 (1 μM) for 4 h or with MVO26630 (50 μM) or BSA (30 mg/ml) for 16 h. Data are the average ± SD of n = 25 microscopic fields for P-STAT3 and n = 14 for LC3II. Statistical significance was calculated using a Dunnett’s multiple comparison test. ns = not significant. (Scale bar = 20μm)

    Journal: Nature Communications

    Article Title: Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis

    doi: 10.1038/s41467-018-07741-6

    Figure Lengend Snippet: Direct STAT3 involvement in regulation of lysosomal hydrolase genes under conditions of protease activity/lysosomal substrate imbalance. a Agarose gel analysis of representative ChIP-PCR products for AEP, CtsD and CtsL promoters in WT and AEP-deficient MEFs. Full data and gel calibration markers can be seen in Supplementary Fig. 6 . b Quantitative PCR analysis of DNA from STAT3 ChIP of WT (blue circles) and AEP-deficient (red squares) MEFs expressed as fold enrichment. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t- test. c AEP activity in WT MEF treated overnight with different concentrations of MVO26630 in the presence (dashed line) or absence (solid line) of 100 μM S3I-201. Data are the average ± SD of three independent experiments. d Quantitative PCR analysis of the mRNA levels for different lysosomal proteases in WT (blue circles) and STAT3c 3T3 (red squares) cells. Data are the average ± SD of n = 6 biologically independent samples for all samples, except for AEP where n = 5 and CtsL where n = 3. Statistical significance was calculated using a two-sided unpaired t- test. e Quantitative PCR analysis of the mRNA levels for different lysosomal proteases in WT MEFs treated (red squares) or not (blue circles) with 30 mg/ml BSA overnight. Data are the average ± SD of n = 4 biologically independent samples for all samples, except for AEP and CtsD where n = 3. Statistical significance was calculated using a two-sided unpaired t- test. f AEP and CtsD/E activities measured in WT and STAT3c 3T3 cells. Data are the average ± SD of n = 3 biologically independent samples. Statistical significance was calculated using a two-sided unpaired t -test. g Activities of AEP, CtsB/L and CtsD/E in WT MEF treated or not with 30 mg/ml BSA overnight in the presence or absence of 100 μM S3I-201. Data are the average ± SD of n = 3 biologically independent samples. h Quantitation of P-STAT3 and LC3II levels in WT MEFs treated with Rapamycin (1 μM) or Torin1 (1 μM) for 4 h or with MVO26630 (50 μM) or BSA (30 mg/ml) for 16 h. Data are the average ± SD of n = 25 microscopic fields for P-STAT3 and n = 14 for LC3II. Statistical significance was calculated using a Dunnett’s multiple comparison test. ns = not significant. (Scale bar = 20μm)

    Article Snippet: Antibodies against Laminin (ab11575), CtsD (ab75852), CtsB (ab92955), Lamp1 (ab24170) and Fibronectin (ab199056) were purchased from AbCam.

    Techniques: Activity Assay, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Quantitation Assay

    CTSL knockdown promoted complementary activation of CTSB, and CTSB overexpression did not affect autophagic clearance. (A to D) 3T3L1 sh Luc (control), as well as sh Ctsl #1 and sh Ctsl #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

    Journal: Autophagy

    Article Title: Involvement of lysosomal dysfunction in autophagosome accumulation and early pathologies in adipose tissue of obese mice

    doi: 10.1080/15548627.2016.1274850

    Figure Lengend Snippet: CTSL knockdown promoted complementary activation of CTSB, and CTSB overexpression did not affect autophagic clearance. (A to D) 3T3L1 sh Luc (control), as well as sh Ctsl #1 and sh Ctsl #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

    Article Snippet: For our Ctsb -overexpression vector, Ctsb cDNA was amplified from a Ctsb vector (Addgene, 11249; deposited by H. Choe) by PCR using PrimeSTAR® HS DNA polymerase (Takara, R010A) and then subcloned into Xho1- and Not1-digested pMXs-AMNN-Puro vector.

    Techniques: Activation Assay, Over Expression, Western Blot, Enzymatic Assay

    Cathepsin B knockdown in OC2 and CAL27 cells reduce cell migration. Western blot analysis showing the cathepsin B Knockdown efficiency in (A) OC2 cell and (B) CAL27 oral cancer cell lines. Detection of cell migration ability by transfected with control siRNA or CTSB siRNA in (C) OC2 cell and (D) CAL27 cell. Results shown that cathepsin B siRNA significantly reduced the migration of OC2 and CAL27 cells. *p

    Journal: PLoS ONE

    Article Title: Cathepsin B Expression and the Correlation with Clinical Aspects of Oral Squamous Cell Carcinoma

    doi: 10.1371/journal.pone.0152165

    Figure Lengend Snippet: Cathepsin B knockdown in OC2 and CAL27 cells reduce cell migration. Western blot analysis showing the cathepsin B Knockdown efficiency in (A) OC2 cell and (B) CAL27 oral cancer cell lines. Detection of cell migration ability by transfected with control siRNA or CTSB siRNA in (C) OC2 cell and (D) CAL27 cell. Results shown that cathepsin B siRNA significantly reduced the migration of OC2 and CAL27 cells. *p

    Article Snippet: Transwell Migration Assays After a treatment with the control siRNA and CTSB siRNA (ID: s3739; Applied Biosystems, Foster City, CA, USA) for 48 h, OC2 and CAL27 cells were harvested and assayed using a Transwell chamber (Millipore Corporation, Billerica, MA, USA) at 7x104 cells/well in serum free medium and then incubated for 48 h at 37°C.

    Techniques: Migration, Western Blot, Transfection

    IFNγ release after iNKT activation in vitro . (A,B) antigen-presenting cells (APCs) (LX2 and C1R-CD1d) were preincubated with or without Cathepsin B (CTSB) and Cathepsin S (CTSS) inhibitors (75 and 7.5 µM, respectively, for 1 h) before being treated with α-GalCer (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the αGC-charged APCs (C1R-CD1d or LX2 cells) with human primary iNKT cells for 24 h, and IFNγ was determined in the extracellular media by enzyme-linked immunosorbent assay (ELISA) (C,D) , APCs (LX2 and C1R-CD1d) were treated with α-GalCer (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the α-GalCer-charged APCs with human primary iNKT cells in the presence or absence of CTSB or CTSS inhibitors for 24 h, and IFNγ was determined in the extracellular media by ELISA. Results are given as a mean ± SD of five independent experiments; * p

    Journal: Frontiers in Immunology

    Article Title: Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion

    doi: 10.3389/fimmu.2018.00391

    Figure Lengend Snippet: IFNγ release after iNKT activation in vitro . (A,B) antigen-presenting cells (APCs) (LX2 and C1R-CD1d) were preincubated with or without Cathepsin B (CTSB) and Cathepsin S (CTSS) inhibitors (75 and 7.5 µM, respectively, for 1 h) before being treated with α-GalCer (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the αGC-charged APCs (C1R-CD1d or LX2 cells) with human primary iNKT cells for 24 h, and IFNγ was determined in the extracellular media by enzyme-linked immunosorbent assay (ELISA) (C,D) , APCs (LX2 and C1R-CD1d) were treated with α-GalCer (100 ng/mL, 6 h). Afterward, we washed APCs and co-cultured the α-GalCer-charged APCs with human primary iNKT cells in the presence or absence of CTSB or CTSS inhibitors for 24 h, and IFNγ was determined in the extracellular media by ELISA. Results are given as a mean ± SD of five independent experiments; * p

    Article Snippet: Medium was supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL, Invitrogen). α-GalCer (Abcam) was given to cells at 100 ng/mL, for 6 h. CTSB inhibitor (75 µM, CA-074 methyl ester, Sigma-Aldrich) and CTSS inhibitor (7.5 µM, Z-FL-COCHO, Calbiochem) were administered 1 h before α-GalCer.

    Techniques: Activation Assay, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

    Effect of Cathepsin B (CTSB) and Cathepsin S (CTSS) inhibitors in liver damage after α-GalCer challenge. Mice were treated with CTSB or CTSS inhibitors (CA-074 or Z-FL, 10 mg/kg, i.p., respectively) 1 h before α-GalCer (2.5 µg/mouse) injection. (A) H E staining of liver samples, arrows indicate inflammatory foci (magnification 20×). Representive images of three independent experiments ( n = 5, each experiment). (B) Liver damage determined by ALT and AST values. * p

    Journal: Frontiers in Immunology

    Article Title: Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion

    doi: 10.3389/fimmu.2018.00391

    Figure Lengend Snippet: Effect of Cathepsin B (CTSB) and Cathepsin S (CTSS) inhibitors in liver damage after α-GalCer challenge. Mice were treated with CTSB or CTSS inhibitors (CA-074 or Z-FL, 10 mg/kg, i.p., respectively) 1 h before α-GalCer (2.5 µg/mouse) injection. (A) H E staining of liver samples, arrows indicate inflammatory foci (magnification 20×). Representive images of three independent experiments ( n = 5, each experiment). (B) Liver damage determined by ALT and AST values. * p

    Article Snippet: Medium was supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL, Invitrogen). α-GalCer (Abcam) was given to cells at 100 ng/mL, for 6 h. CTSB inhibitor (75 µM, CA-074 methyl ester, Sigma-Aldrich) and CTSS inhibitor (7.5 µM, Z-FL-COCHO, Calbiochem) were administered 1 h before α-GalCer.

    Techniques: Mouse Assay, Injection, Staining, AST Assay

    Effect of Cathepsin B (CTSB) and Cathepsin S (CTSS) inhibitors in NKT activation and cytokine secretion after α-GalCer challenge. Mice were treated with CTSB or CTSS inhibitors (CA-074 or Z-FL, 10 mg/kg, i.p., respectively) 1 h before α-GalCer (2.5 µg/mouse) injection. (A) Liver iNKT cells were isolated by liver perfusion, determined by FACS, and activation marker CD69 and apoptotic cell death by Annexin V were determined. (B,C) IFNγ and IL-4 determined by enzyme-linked immunosorbent assay in peripheral blood at the indicated time points. * p

    Journal: Frontiers in Immunology

    Article Title: Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion

    doi: 10.3389/fimmu.2018.00391

    Figure Lengend Snippet: Effect of Cathepsin B (CTSB) and Cathepsin S (CTSS) inhibitors in NKT activation and cytokine secretion after α-GalCer challenge. Mice were treated with CTSB or CTSS inhibitors (CA-074 or Z-FL, 10 mg/kg, i.p., respectively) 1 h before α-GalCer (2.5 µg/mouse) injection. (A) Liver iNKT cells were isolated by liver perfusion, determined by FACS, and activation marker CD69 and apoptotic cell death by Annexin V were determined. (B,C) IFNγ and IL-4 determined by enzyme-linked immunosorbent assay in peripheral blood at the indicated time points. * p

    Article Snippet: Medium was supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL, Invitrogen). α-GalCer (Abcam) was given to cells at 100 ng/mL, for 6 h. CTSB inhibitor (75 µM, CA-074 methyl ester, Sigma-Aldrich) and CTSS inhibitor (7.5 µM, Z-FL-COCHO, Calbiochem) were administered 1 h before α-GalCer.

    Techniques: Activation Assay, Mouse Assay, Injection, Isolation, FACS, Marker, Enzyme-linked Immunosorbent Assay

    Changes in protease gene expression caused by EcTI treatment The EcTI effects on the protease expression in U87 cells were measured by qRT-PCR analysis and compared to non-treated control (c) cells.The effect of EcTI on the expression of the metalloproteases Mmp2 (A) , Mmp9 (B) , Mmp14 (C) , urokinase plasminogen activator, Plau (D) and its receptor, PlauR (E) , Calpain 1, Capn1 (F) , Calpain 2, Capn2 (G) , Cathepsin L, CtsL (H) , and Cathepsin B, CtsB (I) . Error bars indicate SD. * p

    Journal: Oncotarget

    Article Title: Could a plant derived protein potentiate the anticancer effects of a stem cell in brain cancer?

    doi: 10.18632/oncotarget.25090

    Figure Lengend Snippet: Changes in protease gene expression caused by EcTI treatment The EcTI effects on the protease expression in U87 cells were measured by qRT-PCR analysis and compared to non-treated control (c) cells.The effect of EcTI on the expression of the metalloproteases Mmp2 (A) , Mmp9 (B) , Mmp14 (C) , urokinase plasminogen activator, Plau (D) and its receptor, PlauR (E) , Calpain 1, Capn1 (F) , Calpain 2, Capn2 (G) , Cathepsin L, CtsL (H) , and Cathepsin B, CtsB (I) . Error bars indicate SD. * p

    Article Snippet: The real-time PCR reactions were performed using 1:10 dilutions (1 μl/well) of each cDNA sample, TaqMan Universal PCR Master Mix (Applied Biosystems, USA), and TaqMan Gene Expression assays (all from Applied Biosystems, USA) for: Ccnd1 (Cyclin D1), Hs00765553_m1; Cdkn1A (cyclin-dependent kinase inhibitor 1A (p21, Cip1)), Hs00355782_m1; Tp53 (tumor protein p53), Hs01034249_m1; Bax (Bcl-2 associated X protein), Hs99999001_m1; Bcl2 (B-cell CLL/lymphoma2), Hs00608023_m1; Mmp14 (matrix metallopeptidase 14 (membrane-inserted)), Hs00237119_m1; Mmp2 (matrix metallopeptidase 2), Hs1548727_m1; Mmp9 (matrix metallopeptidase 9), Hs00234579_m1; Plaur (plasminogen activator, urokinase receptor), Hs00958880_m1; Plau (plasminogen activator, urokinase), Hs01547054_m1; Capn1 (calpain 1, (mu/l) large subunit), Hs00559804_m1; Capn2 (calpain 2, (m/II) large subunit), Hs00965097_m1; CtsB (cathepsin B), Hs00947433; and CtsL (cathepsin L), Hs00964650_m1.

    Techniques: Expressing, Quantitative RT-PCR

    Sorting, transport and localization of lysosomal proteins in cultured PT ki hepatocytes. A , Isolated hepatocytes from wt and PT ki mice were cultivated for 2 days and the expression of Neu1, Npc2, Ctsl, Ctsb, Ctsd, Ctsz and Lamp1 were determined by Western blotting in the cell lysates and conditioned media. The ER marker protein disulfide isomerase (Pdi) was used as loading control. All soluble lysosomal proteins are missorted to variable degree indicated by reduced intracellular levels and increased secretion into the medium. The intracellular retention of Ctsl, Ctsb and Ctsd are less affected. Comparable Lamp1 levels between wt and PT ki cells are observed. B , Hepatocytes from wt and PT ki mice were labeled with [ 35 S]-methioine for 45 min and either harvested (−, lanes 1, 3 ) or chased for 5 h (+, lanes 2, 4 ) followed by immunoprecipitation of cathepsin D (Ctsd) and cathepsin Z (Ctsz) from cell extracts ( lanes 1–4 ) and media ( lanes 5–6 ). The immunocomplexes were solubilized, separated by SDS-PAGE and visualized by fluorography. Secretion of newly synthesized Ctsd and Ctsz is increased in PT ki hepatocytes, accompanied by a significant reduction or complete loss of processed intracellular mature forms, respectively, during the chase. C , Isolated hepatocytes from wt and PT ki mice were fixed, and immunostained for localization of endogenous cathepsin D ( Ctsd ), cathepsin B ( Ctsb ) and neuraminidase 1 ( Neu1 ) in red and the lysosomal marker protein Lamp1 ( green ). Nuclei were stained with DAPI ( blue ). Only merged images are shown in which yellow indicates colocalization of the antigens. Magnified images of the indicated white rectangles are shown in the insets. Scale bars : 7.5 μm.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II *

    doi: 10.1074/mcp.M116.063636

    Figure Lengend Snippet: Sorting, transport and localization of lysosomal proteins in cultured PT ki hepatocytes. A , Isolated hepatocytes from wt and PT ki mice were cultivated for 2 days and the expression of Neu1, Npc2, Ctsl, Ctsb, Ctsd, Ctsz and Lamp1 were determined by Western blotting in the cell lysates and conditioned media. The ER marker protein disulfide isomerase (Pdi) was used as loading control. All soluble lysosomal proteins are missorted to variable degree indicated by reduced intracellular levels and increased secretion into the medium. The intracellular retention of Ctsl, Ctsb and Ctsd are less affected. Comparable Lamp1 levels between wt and PT ki cells are observed. B , Hepatocytes from wt and PT ki mice were labeled with [ 35 S]-methioine for 45 min and either harvested (−, lanes 1, 3 ) or chased for 5 h (+, lanes 2, 4 ) followed by immunoprecipitation of cathepsin D (Ctsd) and cathepsin Z (Ctsz) from cell extracts ( lanes 1–4 ) and media ( lanes 5–6 ). The immunocomplexes were solubilized, separated by SDS-PAGE and visualized by fluorography. Secretion of newly synthesized Ctsd and Ctsz is increased in PT ki hepatocytes, accompanied by a significant reduction or complete loss of processed intracellular mature forms, respectively, during the chase. C , Isolated hepatocytes from wt and PT ki mice were fixed, and immunostained for localization of endogenous cathepsin D ( Ctsd ), cathepsin B ( Ctsb ) and neuraminidase 1 ( Neu1 ) in red and the lysosomal marker protein Lamp1 ( green ). Nuclei were stained with DAPI ( blue ). Only merged images are shown in which yellow indicates colocalization of the antigens. Magnified images of the indicated white rectangles are shown in the insets. Scale bars : 7.5 μm.

    Article Snippet: Ctsb was purchased from Neuromics, Ctsz, and Ctsl from R & D Systems (Minneapolis, MN), Lrp1 and Ldlr from Abcam.

    Techniques: Cell Culture, Isolation, Mouse Assay, Expressing, Western Blot, Marker, Labeling, Immunoprecipitation, SDS Page, Synthesized, Staining

    Analysis of lysosomal proteins that are specifically changed in their expression in PT ki lysosomes. Western blotting analysis for Neu1, Npc2, Ctsl, Ctsh, Ctsa, Ctsb, Ctsd, Ctsz, Gusb, Ctss, and Lamp1 in lysosome-enriched fractions from wt and PT ki mice. Densitometric evaluation of the corresponding immunoreactive bands confirms the significant differences between wt and PT ki samples ( n = 4; sc , single chain; hc , heavy chain; lc , light chain; ns , not significant; p

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II *

    doi: 10.1074/mcp.M116.063636

    Figure Lengend Snippet: Analysis of lysosomal proteins that are specifically changed in their expression in PT ki lysosomes. Western blotting analysis for Neu1, Npc2, Ctsl, Ctsh, Ctsa, Ctsb, Ctsd, Ctsz, Gusb, Ctss, and Lamp1 in lysosome-enriched fractions from wt and PT ki mice. Densitometric evaluation of the corresponding immunoreactive bands confirms the significant differences between wt and PT ki samples ( n = 4; sc , single chain; hc , heavy chain; lc , light chain; ns , not significant; p

    Article Snippet: Ctsb was purchased from Neuromics, Ctsz, and Ctsl from R & D Systems (Minneapolis, MN), Lrp1 and Ldlr from Abcam.

    Techniques: Expressing, Western Blot, Mouse Assay

    Predominantly cytosolic TFEB and abnormal levels of phosphorylated TFE 3 in RPE cells from fasted and fed Cryba1 KO mice. (A) Western blot showing increased levels of TFEB in Cryba1 fl/fl nuclear extracts following fasting. TFEB is predominantly localized in the cytosol (C) in cells from normally fed Cryba1 fl/fl (control) mice, but after fasting the nuclear (N) proportion increases. In cells from Cryba1 KO mice, significantly lower nuclear levels of TFEB were observed in both fasted and fed conditions. (B) Cryba1 fl/fl and Cryba1 KO cells were subjected to qPCR analysis using Taqman probes for some CLEAR (coordinated lysosomal expression and regulation) genes after fasting. Cryba1 KO cells showed a reduction in the transcript levels for lysosomal hydrolases: CTSD (55%), CTSB (75%); lysosomal acidification: ATP 6V0A1 (56%); lysosomal membrane proteins: LAMP 1 (15%), LAMP 2 (40%), and MCOLIN 1 (35%); and gene related to autophagy: BECN 1 (51%), UVRAG (35%). The graph shows mean ± SD from triplicate experiments, representative of at least three independent experiments. (two‐tailed t‐test) (C) Western blot for p‐ TFE 3 in RPE isolated from Cryba1 fl/fl and Cryba1 KO mice. The data indicate decreased p‐ TFE 3 in controls, but increased p‐ TFE 3 in KO RPE after fasting. (n = 4) Quantification of C is shown in D. (E) Western blot showing cathepsin D (both precursor and immature forms) expression in the RPE from four‐month‐old (n = 6) and 10‐month‐old (n = 3) Cryba1 KO mice vs. floxed controls. F and G show densitometric quantification for data in E. (H) CTSD immunostaining (red) and DAPI staining (blue) on RPE flat mounts from Cryba1 fl/fl and Cryba1 KO mice at 2 and 10 months of age (n = 3). There are fewer fluorescent puncta in Cryba1 KO RPE cells, than in Cryba1 fl/fl RPE at both ages. Scale bars=10 μm. (I) TEM of RPE in 20‐month‐old Cryba1 fl/fl mouse showing photoreceptor outer segments ( POS ) and RPE (left panel). Unlike Cryba1 KO mouse, TEM (center panel) showed numerous vacuoles with possible accumulation of lipid‐like droplets (arrow). TEM also showed many autolysosomes, some with incomplete degradation (arrow) retained in 20‐month‐old Cryba1 KO mouse (right panel). (J) Western blot and quantification (K) of protein level of SQSTM 1 (p62) in RPE of 10‐month‐old Cryba1 KO mice relative to control mice. (n = 3) * P

    Journal: Aging Cell

    Article Title: The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling

    doi: 10.1111/acel.12561

    Figure Lengend Snippet: Predominantly cytosolic TFEB and abnormal levels of phosphorylated TFE 3 in RPE cells from fasted and fed Cryba1 KO mice. (A) Western blot showing increased levels of TFEB in Cryba1 fl/fl nuclear extracts following fasting. TFEB is predominantly localized in the cytosol (C) in cells from normally fed Cryba1 fl/fl (control) mice, but after fasting the nuclear (N) proportion increases. In cells from Cryba1 KO mice, significantly lower nuclear levels of TFEB were observed in both fasted and fed conditions. (B) Cryba1 fl/fl and Cryba1 KO cells were subjected to qPCR analysis using Taqman probes for some CLEAR (coordinated lysosomal expression and regulation) genes after fasting. Cryba1 KO cells showed a reduction in the transcript levels for lysosomal hydrolases: CTSD (55%), CTSB (75%); lysosomal acidification: ATP 6V0A1 (56%); lysosomal membrane proteins: LAMP 1 (15%), LAMP 2 (40%), and MCOLIN 1 (35%); and gene related to autophagy: BECN 1 (51%), UVRAG (35%). The graph shows mean ± SD from triplicate experiments, representative of at least three independent experiments. (two‐tailed t‐test) (C) Western blot for p‐ TFE 3 in RPE isolated from Cryba1 fl/fl and Cryba1 KO mice. The data indicate decreased p‐ TFE 3 in controls, but increased p‐ TFE 3 in KO RPE after fasting. (n = 4) Quantification of C is shown in D. (E) Western blot showing cathepsin D (both precursor and immature forms) expression in the RPE from four‐month‐old (n = 6) and 10‐month‐old (n = 3) Cryba1 KO mice vs. floxed controls. F and G show densitometric quantification for data in E. (H) CTSD immunostaining (red) and DAPI staining (blue) on RPE flat mounts from Cryba1 fl/fl and Cryba1 KO mice at 2 and 10 months of age (n = 3). There are fewer fluorescent puncta in Cryba1 KO RPE cells, than in Cryba1 fl/fl RPE at both ages. Scale bars=10 μm. (I) TEM of RPE in 20‐month‐old Cryba1 fl/fl mouse showing photoreceptor outer segments ( POS ) and RPE (left panel). Unlike Cryba1 KO mouse, TEM (center panel) showed numerous vacuoles with possible accumulation of lipid‐like droplets (arrow). TEM also showed many autolysosomes, some with incomplete degradation (arrow) retained in 20‐month‐old Cryba1 KO mouse (right panel). (J) Western blot and quantification (K) of protein level of SQSTM 1 (p62) in RPE of 10‐month‐old Cryba1 KO mice relative to control mice. (n = 3) * P

    Article Snippet: PCR amplification was performed using the 7500 PCR Fast Real‐Time System (Applied Biosystems, Carlsbad, CA, USA) and custom‐made TaqMan probes for LAMP1 (Mm00495262_m1), LAMP2A (Mm00495274_m1), cathepsin B (Mm01310506_m1), cathepsin D (Mm00515586_m1), V‐ATPase (Mm00724370_m1), UVRAG (Mm00724370_m1), Beclin‐1 (Mm01265461_m1), and Mucolipin‐1 (Mm00522549_m1).

    Techniques: Mouse Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test, Isolation, Immunostaining, Staining, Transmission Electron Microscopy