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    Millipore cathepsin k inhibitor ii
    Effect of <t>cathepsin</t> K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P
    Cathepsin K Inhibitor Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of cathepsin K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Effect of cathepsin K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Isolation, Mouse Assay

    Echocardiographic features in wild-type (WT) and cathepsin K knockout mice fed normal diet or high-fat diet. A : Left ventricular wall thickness. B : LVEDD. C : LVESD. D : Fraction shortening [(LVEDD − LVESD) / LVEDD × 100]. Mean ± SEM, n = 6–7 mice per group, * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Echocardiographic features in wild-type (WT) and cathepsin K knockout mice fed normal diet or high-fat diet. A : Left ventricular wall thickness. B : LVEDD. C : LVESD. D : Fraction shortening [(LVEDD − LVESD) / LVEDD × 100]. Mean ± SEM, n = 6–7 mice per group, * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay

    A and B : Effect of cathepsin K knockout on glucose disposal in mice fed a normal diet (ND) or a high-fat diet (HFD) after an intraperitoneal glucose (2 g/kg, body weight) challenge ( A ). Integrated area under the postglucose challenge, glucose-disposal curve ( B ). C : Effect of ND and HFD, respectively, on body weight gain over a 20-week period in wild-type and cathepsin K knockout mice. D : Representative images of wild-type (WT) and cathepsin K knockout mice fed ND or HFD and representative hearts from each group. E–H : Effect of cathepsin K knockout on HFD-induced cardiac hypertrophy. Representative images using wheat germ agglutinin staining of the left ventricular tissue ( E ), quantitation of cardiomyocyte cross-sectional area ( F ), and representative gel blots for GATA4, NFATc3, ANP, and GAPDH (loading control) ( G ), and size of isolated cardiomyocytes ( H ). Data are represented as mean ± SEM, n = 5–7 mice per group, * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: A and B : Effect of cathepsin K knockout on glucose disposal in mice fed a normal diet (ND) or a high-fat diet (HFD) after an intraperitoneal glucose (2 g/kg, body weight) challenge ( A ). Integrated area under the postglucose challenge, glucose-disposal curve ( B ). C : Effect of ND and HFD, respectively, on body weight gain over a 20-week period in wild-type and cathepsin K knockout mice. D : Representative images of wild-type (WT) and cathepsin K knockout mice fed ND or HFD and representative hearts from each group. E–H : Effect of cathepsin K knockout on HFD-induced cardiac hypertrophy. Representative images using wheat germ agglutinin staining of the left ventricular tissue ( E ), quantitation of cardiomyocyte cross-sectional area ( F ), and representative gel blots for GATA4, NFATc3, ANP, and GAPDH (loading control) ( G ), and size of isolated cardiomyocytes ( H ). Data are represented as mean ± SEM, n = 5–7 mice per group, * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay, Staining, Quantitation Assay, Aqueous Normal-phase Chromatography, Isolation

    Cardiomyocyte contractile properties in wild-type (WT) and cathepsin K knockout mice fed normal diet (ND) or high-fat diet (HFD). A and B : Representative traces from cardiomyocytes isolated from wild-type and cathepsin K knockout mice. C–H : Resting cell length, peak shortening (normalized to cell length), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt), time-to-peak shortening (TPS), and time to 90% relengthening (TR 90 ), respectively. Mean ± SEM, n = 76–87 cells per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Cardiomyocyte contractile properties in wild-type (WT) and cathepsin K knockout mice fed normal diet (ND) or high-fat diet (HFD). A and B : Representative traces from cardiomyocytes isolated from wild-type and cathepsin K knockout mice. C–H : Resting cell length, peak shortening (normalized to cell length), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt), time-to-peak shortening (TPS), and time to 90% relengthening (TR 90 ), respectively. Mean ± SEM, n = 76–87 cells per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay, Isolation

    Intracellular Ca 2+ transients in cardiomyocytes and expression levels of proteins related to intracellular Ca 2+ handling in wild-type (WT) and cathepsin K knockout mice fed a normal diet (ND) or high-fat diet (HFD). A–D : Resting fura-2 fluorescence intensity (FFI), electrically stimulated increase in FFI (ΔFFI), single exponential intracellular Ca 2+ decay, and peak FFI, respectively. E : Representative Western blot images for phospho-phospholamban, phospholamban (PLB), SERCA2, and GAPDH (loading control). F–H : Densitometric quantitation of SERCA normalized to GAPDH, PLB normalized to GAPDH, and PBL-to-SERCA ratio, respectively. Mean ± SEM, n = 94–100 cells or 5–7 mice per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Intracellular Ca 2+ transients in cardiomyocytes and expression levels of proteins related to intracellular Ca 2+ handling in wild-type (WT) and cathepsin K knockout mice fed a normal diet (ND) or high-fat diet (HFD). A–D : Resting fura-2 fluorescence intensity (FFI), electrically stimulated increase in FFI (ΔFFI), single exponential intracellular Ca 2+ decay, and peak FFI, respectively. E : Representative Western blot images for phospho-phospholamban, phospholamban (PLB), SERCA2, and GAPDH (loading control). F–H : Densitometric quantitation of SERCA normalized to GAPDH, PLB normalized to GAPDH, and PBL-to-SERCA ratio, respectively. Mean ± SEM, n = 94–100 cells or 5–7 mice per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Expressing, Knock-Out, Mouse Assay, Fluorescence, Western Blot, Quantitation Assay

    A : Immunolocalization of cathepsin K in cultured cells. H9c2 myoblasts were labeled using lysosomal dye and fluorogenic substrate MR-(LR) 2 , which detects active cathepsin K. Upper panel (merged figure) shows colocalization of cathepsin K in lysosomes under basal conditions. On stimulation with palmitic acid, active cathepsin K was released from lysosomes to the cytoplasm (indicated by arrows). B–E . Effect of cathepsin K knockout on cardiac insulin signaling molecules in normal diet (ND)-fed and high-fat diet (HFD)-fed mice. Mice were challenged with insulin (1.5 U/100 g body weight, intraperitoneally) for 10 min before isolation of the heart tissue for Western blot. Insulin-stimulated phosphorylation of Akt (indicated by an asterisk), total Akt, basal phospho-Akt, basal levels of insulin receptor-β, and glucose transporter-4 (Glut4) were assessed by Western blotting ( B ) and quantitated by densitometry ( C–E ). F and G : Effect of cathepsin K knockout on the protein levels of other cathepsins in ND-fed and HFD-fed mice. Mean ± SEM, n = 3–4 per group. WT, wild-type. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: A : Immunolocalization of cathepsin K in cultured cells. H9c2 myoblasts were labeled using lysosomal dye and fluorogenic substrate MR-(LR) 2 , which detects active cathepsin K. Upper panel (merged figure) shows colocalization of cathepsin K in lysosomes under basal conditions. On stimulation with palmitic acid, active cathepsin K was released from lysosomes to the cytoplasm (indicated by arrows). B–E . Effect of cathepsin K knockout on cardiac insulin signaling molecules in normal diet (ND)-fed and high-fat diet (HFD)-fed mice. Mice were challenged with insulin (1.5 U/100 g body weight, intraperitoneally) for 10 min before isolation of the heart tissue for Western blot. Insulin-stimulated phosphorylation of Akt (indicated by an asterisk), total Akt, basal phospho-Akt, basal levels of insulin receptor-β, and glucose transporter-4 (Glut4) were assessed by Western blotting ( B ) and quantitated by densitometry ( C–E ). F and G : Effect of cathepsin K knockout on the protein levels of other cathepsins in ND-fed and HFD-fed mice. Mean ± SEM, n = 3–4 per group. WT, wild-type. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Cell Culture, Labeling, Knock-Out, Mouse Assay, Isolation, Western Blot

    PGRN processing into GRNs is mediated by proper lysosome function and cysteine protease activity. A , HAP1 WT cells were treated for 24 h with the pan-lysosome inhibitors chloroquine (CQ; 50 µM), BafA1 (50 nM), or concanamycin A (ConA; 50 nM) and conditioned media were analyzed for secreted PGRN by ELISA. B , Lysates from HAP1 WT cells treated as in A were analyzed for PGRN and GRN-2,3 by immunoblot. C , D , Quantification of ( C ) PGRN and ( D ) GRN-2,3 from the experiment in B . E , HAP1 WT cells were treated with the indicated protease inhibitors for 24 h and analyzed for PGRN and GRN-2,3 by immunoblot. The inhibitors, their primary targets, and concentrations used are shown in the table at right. F , Time-dependent cleavage of C-TAP PGRN by recombinant cathepsin L in vitro . G , C-TAP PGRN was incubated with or without cathepsin L for 2 h in vitro and analyzed for multiple GRNs by immunoblot. H , HAP1 WT cells were treated with increasing concentrations of cathepsin L inhibitor II (Z-FY-CHO) for 40 h and lysates were analyzed for PGRN and GRN-2,3 by immunoblot. I , J , Quantification of ( I ) PGRN and ( J ) GRN-2,3 from the experiment in H . B , E , H , Arrows denote endogenous, intermediate PGRN cleavage products. For all immunoblots, PGRN and GRN-2,3 were detected with R D AF2420 and Sigma antibodies respectively. All immunoblot images are representative of at least three independent experiments and quantitative data are presented as mean ± SEM of three independent experiments; *differs from control p

    Journal: eNeuro

    Article Title: Intracellular Proteolysis of Progranulin Generates Stable, Lysosomal Granulins that Are Haploinsufficient in Patients with Frontotemporal Dementia Caused by GRN Mutations

    doi: 10.1523/ENEURO.0100-17.2017

    Figure Lengend Snippet: PGRN processing into GRNs is mediated by proper lysosome function and cysteine protease activity. A , HAP1 WT cells were treated for 24 h with the pan-lysosome inhibitors chloroquine (CQ; 50 µM), BafA1 (50 nM), or concanamycin A (ConA; 50 nM) and conditioned media were analyzed for secreted PGRN by ELISA. B , Lysates from HAP1 WT cells treated as in A were analyzed for PGRN and GRN-2,3 by immunoblot. C , D , Quantification of ( C ) PGRN and ( D ) GRN-2,3 from the experiment in B . E , HAP1 WT cells were treated with the indicated protease inhibitors for 24 h and analyzed for PGRN and GRN-2,3 by immunoblot. The inhibitors, their primary targets, and concentrations used are shown in the table at right. F , Time-dependent cleavage of C-TAP PGRN by recombinant cathepsin L in vitro . G , C-TAP PGRN was incubated with or without cathepsin L for 2 h in vitro and analyzed for multiple GRNs by immunoblot. H , HAP1 WT cells were treated with increasing concentrations of cathepsin L inhibitor II (Z-FY-CHO) for 40 h and lysates were analyzed for PGRN and GRN-2,3 by immunoblot. I , J , Quantification of ( I ) PGRN and ( J ) GRN-2,3 from the experiment in H . B , E , H , Arrows denote endogenous, intermediate PGRN cleavage products. For all immunoblots, PGRN and GRN-2,3 were detected with R D AF2420 and Sigma antibodies respectively. All immunoblot images are representative of at least three independent experiments and quantitative data are presented as mean ± SEM of three independent experiments; *differs from control p

    Article Snippet: Cathepsin L inhibitor II (Z-FY-CHO) and ALLN were from Calbiochem.

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Recombinant, In Vitro, Incubation, Western Blot

    Model of intracellular processing of PGRN into stable, lysosomal GRNs. Sortilin and other receptors target endocytosed and newly synthesized PGRN to lysosomes. Within lysosomes, PGRN is proteolytically cleaved, in part, by cysteine proteases (i.e., cathepsin L) into mature, stable GRN proteins. Ablation of sortilin results in the reduced production of GRNs. Further, lysosome dysfunction caused by alkalizing agents or TMEM106B overexpression inhibits the processing of PGRN into GRNs. FTD- GRN  patients are haploinsufficient for GRNs, which may drive lysosome dysfunction leading to neurodegeneration.

    Journal: eNeuro

    Article Title: Intracellular Proteolysis of Progranulin Generates Stable, Lysosomal Granulins that Are Haploinsufficient in Patients with Frontotemporal Dementia Caused by GRN Mutations

    doi: 10.1523/ENEURO.0100-17.2017

    Figure Lengend Snippet: Model of intracellular processing of PGRN into stable, lysosomal GRNs. Sortilin and other receptors target endocytosed and newly synthesized PGRN to lysosomes. Within lysosomes, PGRN is proteolytically cleaved, in part, by cysteine proteases (i.e., cathepsin L) into mature, stable GRN proteins. Ablation of sortilin results in the reduced production of GRNs. Further, lysosome dysfunction caused by alkalizing agents or TMEM106B overexpression inhibits the processing of PGRN into GRNs. FTD- GRN patients are haploinsufficient for GRNs, which may drive lysosome dysfunction leading to neurodegeneration.

    Article Snippet: Cathepsin L inhibitor II (Z-FY-CHO) and ALLN were from Calbiochem.

    Techniques: Synthesized, Over Expression

    Cathepsins act upstream of TGF- β 1 and UVR stimulates FAP- α -dependent invasion. ( A and B ) Primary melanoma cells (FM55P, WM115, WM278, WM793) were irradiated with UVA (6 J cm −2 ) or UVB (60 mJcm −2 ) and when indicated, treated with inhibitors of cathepsin B (CA-074 Me; 1 μ M ), anti-cathepsin D (anti-c at D; 42 μ g ml −1 ), cathepsin K (Inh II; 10 μ M ). The conditioned media were collected. ( A ) TGF- β 1 release to the medium was analysed by immunoblot. Samples were collected from equal number of cells and representative blots are shown (mean±s.d.; n =4; * P

    Journal: British Journal of Cancer

    Article Title: UV radiation promotes melanoma dissemination mediated by the sequential reaction axis of cathepsins–TGF-β1–FAP-α

    doi: 10.1038/bjc.2017.182

    Figure Lengend Snippet: Cathepsins act upstream of TGF- β 1 and UVR stimulates FAP- α -dependent invasion. ( A and B ) Primary melanoma cells (FM55P, WM115, WM278, WM793) were irradiated with UVA (6 J cm −2 ) or UVB (60 mJcm −2 ) and when indicated, treated with inhibitors of cathepsin B (CA-074 Me; 1 μ M ), anti-cathepsin D (anti-c at D; 42 μ g ml −1 ), cathepsin K (Inh II; 10 μ M ). The conditioned media were collected. ( A ) TGF- β 1 release to the medium was analysed by immunoblot. Samples were collected from equal number of cells and representative blots are shown (mean±s.d.; n =4; * P

    Article Snippet: Inhibitors of cathepsin B (CA-074 Me; 1 μ M ) and cathepsin K (inhibitor II; 10 μ M ) were from Calbiochem (San Diego, CA, US) and preincubated for 16 h. Lysosomal acidity was neutralised by NH4 Cl (10 mM , 30 min, Sigma, St Louis, MO, USA).

    Techniques: Activated Clotting Time Assay, Irradiation

    RNF41 blocks formation of the LR CTS and concomitantly enhances LR shedding. ( A ) HEK293T cells were cotransfected with hLR–HA and increasing amounts of Etag–RNF41 or Etag–RNF41 ΔRING. After overnight chloroquine incubation, cell lysates were analysed using western blotting (WB). ( B ) HEK293T cells were cotransfected with mLRsh–FLAG and Etag–RNF41 or Etag–RNF41 ΔRING. After overnight chloroquine incubation, cell lysates were analysed using western blotting (WB). FL: full length; CTS: C-terminal stub. ( C ) Analysis of procathepsin L (42 kDa) and cathepsin L (34 and 26 kDa) expression after RNF41 (ΔRING) expression. ( D ) Cell media supernatants from the transfectants in Fig. 5A were analysed for soluble LR levels. ( E ) RNF41-enhanced LR shedding is reversed by TAPI-1. HEK293T cells transfected with hLR–HA with or without full length Etag–RNF41 were incubated overnight in starvation medium with TAPI-1 or cathepsin L inhibitor III and soluble LR levels in the cell media supernatants were quantified. ( F ) RNF41-enhanced LR shedding is reversed by silencing of ADAM10 and ADAM17 . HEK293T cells, reverse transfected with siRNA targeting ADAM10 or ADAM17 , were cotransfected the next day with hLR–HA with or without full-length Etag–RNF41 and soluble LR levels in the cell media supernatants were quantified. Values are means ± s.d. ( n =3). * P

    Journal: Journal of Cell Science

    Article Title: RNF41 (Nrdp1) controls type 1 cytokine receptor degradation and ectodomain shedding

    doi: 10.1242/jcs.078055

    Figure Lengend Snippet: RNF41 blocks formation of the LR CTS and concomitantly enhances LR shedding. ( A ) HEK293T cells were cotransfected with hLR–HA and increasing amounts of Etag–RNF41 or Etag–RNF41 ΔRING. After overnight chloroquine incubation, cell lysates were analysed using western blotting (WB). ( B ) HEK293T cells were cotransfected with mLRsh–FLAG and Etag–RNF41 or Etag–RNF41 ΔRING. After overnight chloroquine incubation, cell lysates were analysed using western blotting (WB). FL: full length; CTS: C-terminal stub. ( C ) Analysis of procathepsin L (42 kDa) and cathepsin L (34 and 26 kDa) expression after RNF41 (ΔRING) expression. ( D ) Cell media supernatants from the transfectants in Fig. 5A were analysed for soluble LR levels. ( E ) RNF41-enhanced LR shedding is reversed by TAPI-1. HEK293T cells transfected with hLR–HA with or without full length Etag–RNF41 were incubated overnight in starvation medium with TAPI-1 or cathepsin L inhibitor III and soluble LR levels in the cell media supernatants were quantified. ( F ) RNF41-enhanced LR shedding is reversed by silencing of ADAM10 and ADAM17 . HEK293T cells, reverse transfected with siRNA targeting ADAM10 or ADAM17 , were cotransfected the next day with hLR–HA with or without full-length Etag–RNF41 and soluble LR levels in the cell media supernatants were quantified. Values are means ± s.d. ( n =3). * P

    Article Snippet: One day (or 8 hours for confocal imaging) after transfection, cells were left untreated (DMSO) or treated overnight with chloroquine (25 μM), phorbol 12-myristate 13-acetate (PMA) (1 μg/ml), N-ethylmaleimide (NEM) (5 nM), E-64d (10 μM), pepstatin A (10 μM), z-FA-fmk (10 μM) (Sigma), TAPI-1 (50 μM) (Peptides International), Brefeldin A (BFA) (5 μg/ml), calpeptin, phenylmethylsulfonyl fluoride (PMSF), leupeptin, GM6001, cathepsin L inhibitor II/III/IV, cathepsin K inhibitor II, cathepsin S inhibitor, CA-074 Me (all at 10 μM) (Calbiochem).

    Techniques: Incubation, Western Blot, Expressing, Transfection

    The LR is cleaved by cathepsin L. Cell lysates of HEK293T cells transiently expressing the long isoform of the human LR with a C-terminal HA-tag (hLR–HA) or the short isoform of the mouse LR with a C-terminal FLAG-tag (mLRsh–FLAG) were analysed using western blotting (WB). ( A ) Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine, in the absence of stimulus or in combination with leptin for 0.5 or 24 hours. ( B ) Cells expressing mLRsh–FLAG were left untreated (DMSO) or were incubated overnight with chloroquine. ( C , D ) Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine alone or together with the indicated protease inhibitors. ( E ) Cells expressing mLRsh–FLAG were left untreated (DMSO) or were incubated overnight with chloroquine alone or together with cathepsin L inhibitor III. ( F ) The remaining C-terminal fragment is membrane anchored. Untransfected HEK293T cells or HEK293T cells transiently expressing hLR–HA were incubated overnight with chloroquine. Cell homogenates were separated in a nuclear (NUC), cytoplasmic (CYT), membrane (MEM) and rest fraction by differential centrifugation and analysed for the presence of the FL hLR (top square) or the CTS (bottom square) by western blotting (WB). M, molecular weight marker. ( G ) Activation or inhibition of ectodomain shedding has no effect on LR cleavage by cathepsin L. Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine alone or in combination with calpeptin, cathepsin L inhibitor III, NEM or PMA with or without TAPI-1. ( H ) Brefeldin A (BFA) incubation blocks LR-CTS formation. Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine alone or in combination with BFA. FL, full length; CTS, C-terminal stub.

    Journal: Journal of Cell Science

    Article Title: RNF41 (Nrdp1) controls type 1 cytokine receptor degradation and ectodomain shedding

    doi: 10.1242/jcs.078055

    Figure Lengend Snippet: The LR is cleaved by cathepsin L. Cell lysates of HEK293T cells transiently expressing the long isoform of the human LR with a C-terminal HA-tag (hLR–HA) or the short isoform of the mouse LR with a C-terminal FLAG-tag (mLRsh–FLAG) were analysed using western blotting (WB). ( A ) Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine, in the absence of stimulus or in combination with leptin for 0.5 or 24 hours. ( B ) Cells expressing mLRsh–FLAG were left untreated (DMSO) or were incubated overnight with chloroquine. ( C , D ) Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine alone or together with the indicated protease inhibitors. ( E ) Cells expressing mLRsh–FLAG were left untreated (DMSO) or were incubated overnight with chloroquine alone or together with cathepsin L inhibitor III. ( F ) The remaining C-terminal fragment is membrane anchored. Untransfected HEK293T cells or HEK293T cells transiently expressing hLR–HA were incubated overnight with chloroquine. Cell homogenates were separated in a nuclear (NUC), cytoplasmic (CYT), membrane (MEM) and rest fraction by differential centrifugation and analysed for the presence of the FL hLR (top square) or the CTS (bottom square) by western blotting (WB). M, molecular weight marker. ( G ) Activation or inhibition of ectodomain shedding has no effect on LR cleavage by cathepsin L. Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine alone or in combination with calpeptin, cathepsin L inhibitor III, NEM or PMA with or without TAPI-1. ( H ) Brefeldin A (BFA) incubation blocks LR-CTS formation. Cells expressing hLR–HA were left untreated (DMSO) or were incubated overnight with chloroquine alone or in combination with BFA. FL, full length; CTS, C-terminal stub.

    Article Snippet: One day (or 8 hours for confocal imaging) after transfection, cells were left untreated (DMSO) or treated overnight with chloroquine (25 μM), phorbol 12-myristate 13-acetate (PMA) (1 μg/ml), N-ethylmaleimide (NEM) (5 nM), E-64d (10 μM), pepstatin A (10 μM), z-FA-fmk (10 μM) (Sigma), TAPI-1 (50 μM) (Peptides International), Brefeldin A (BFA) (5 μg/ml), calpeptin, phenylmethylsulfonyl fluoride (PMSF), leupeptin, GM6001, cathepsin L inhibitor II/III/IV, cathepsin K inhibitor II, cathepsin S inhibitor, CA-074 Me (all at 10 μM) (Calbiochem).

    Techniques: Expressing, FLAG-tag, Western Blot, Incubation, Centrifugation, Molecular Weight, Marker, Activation Assay, Inhibition

    Cathepsin activity and cystatin C amount in primary and metastatic human melanoma cells . ( A ) Quantitative cytofluorimetric analysis of plasma membrane cathepsin B, D and L in four different primary (white columns) and metastatic (black columns) melanoma cell lines. Numbers represent the mean values among four different primary or metastatic melanoma cell lines analyzed. ( B ) Fluorimetric assays for cathepsin B (black columns), cathepsin D (grey columns) and cathepsin L (white columns) activity in the cell medium of two representative primary and metastatic melanoma cell lines. Results are reported as fluorescence units. ( C ) Concentration of cystatin C in the growth medium of two representative primary and metastatic melanoma cell lines obtained by ELISA test. Results are reported as ng/ml. (*) p

    Journal: Molecular Cancer

    Article Title: Cathepsin B inhibition interferes with metastatic potential of human melanoma: an in vitro and in vivo study

    doi: 10.1186/1476-4598-9-207

    Figure Lengend Snippet: Cathepsin activity and cystatin C amount in primary and metastatic human melanoma cells . ( A ) Quantitative cytofluorimetric analysis of plasma membrane cathepsin B, D and L in four different primary (white columns) and metastatic (black columns) melanoma cell lines. Numbers represent the mean values among four different primary or metastatic melanoma cell lines analyzed. ( B ) Fluorimetric assays for cathepsin B (black columns), cathepsin D (grey columns) and cathepsin L (white columns) activity in the cell medium of two representative primary and metastatic melanoma cell lines. Results are reported as fluorescence units. ( C ) Concentration of cystatin C in the growth medium of two representative primary and metastatic melanoma cell lines obtained by ELISA test. Results are reported as ng/ml. (*) p

    Article Snippet: For cell migration and invasion assays, cells were seeded in the presence of the cathepsin D inhibitor (Pepstatin A, 100 μM, Calbiochem), the cathepsin L inhibitor (cathepsin L inhibitor II, Z-FY-CHO, 10 μM, Calbiochem) as well as two cathepsin B inhibitors: CA-074 (cell unpermeant) and CA-074Me (cell permeant) (both given at a concentration of 10 μM, Calbiochem, Nottingham, UK).

    Techniques: Activity Assay, Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Cathepsins and in vitro invasiveness: effect of chemical and biological inhibitors . ( A ) Invasion test on primary (white columns) and metastatic (black columns) melanoma cell lines. ( B ) Invasion test on two representative cell lines from primary (white columns) and metastatic (black columns) melanoma in the presence of CA-074 (left panel), Pepstatin A (central panel) or cathepsin L inhibitor (right panel). Dotted lines represent the mean of the results obtained in primary melanoma cell lines, and dashed lines indicate the mean of the results obtained in metastatic melanoma cell lines by using DMSO (vehicle of the cathepsin inhibitors). ( C ) Invasion test on two representative cell lines from primary (white columns) and metastatic (black columns) melanoma in the absence or in the presence of specific antibodies against cathepsin B (left panel), cathepsin D (central panel) or cathepsin L (right panel). Dotted lines represent the mean of the results obtained in primary melanoma cell lines, and dashed lines indicate the mean of the results obtained in metastatic melanoma cell lines by using IgG1 as positive control. ( D ) Histogram showing the results obtained from four different primary melanoma (white columns) or metastatic melanoma (black columns) cell lines in the absence or in the presence of CA-074 or antibodies against cathepsin B. Data are reported as mean ± SD of the percentage of invading cells. Student's t -test indicates: p = 0.0097 for untreated MM cells vs. CA-074-treated MM cells and p = 0.0030 for untreated MM cells vs. MM cells treated with anti-cathepsin B antibodies.

    Journal: Molecular Cancer

    Article Title: Cathepsin B inhibition interferes with metastatic potential of human melanoma: an in vitro and in vivo study

    doi: 10.1186/1476-4598-9-207

    Figure Lengend Snippet: Cathepsins and in vitro invasiveness: effect of chemical and biological inhibitors . ( A ) Invasion test on primary (white columns) and metastatic (black columns) melanoma cell lines. ( B ) Invasion test on two representative cell lines from primary (white columns) and metastatic (black columns) melanoma in the presence of CA-074 (left panel), Pepstatin A (central panel) or cathepsin L inhibitor (right panel). Dotted lines represent the mean of the results obtained in primary melanoma cell lines, and dashed lines indicate the mean of the results obtained in metastatic melanoma cell lines by using DMSO (vehicle of the cathepsin inhibitors). ( C ) Invasion test on two representative cell lines from primary (white columns) and metastatic (black columns) melanoma in the absence or in the presence of specific antibodies against cathepsin B (left panel), cathepsin D (central panel) or cathepsin L (right panel). Dotted lines represent the mean of the results obtained in primary melanoma cell lines, and dashed lines indicate the mean of the results obtained in metastatic melanoma cell lines by using IgG1 as positive control. ( D ) Histogram showing the results obtained from four different primary melanoma (white columns) or metastatic melanoma (black columns) cell lines in the absence or in the presence of CA-074 or antibodies against cathepsin B. Data are reported as mean ± SD of the percentage of invading cells. Student's t -test indicates: p = 0.0097 for untreated MM cells vs. CA-074-treated MM cells and p = 0.0030 for untreated MM cells vs. MM cells treated with anti-cathepsin B antibodies.

    Article Snippet: For cell migration and invasion assays, cells were seeded in the presence of the cathepsin D inhibitor (Pepstatin A, 100 μM, Calbiochem), the cathepsin L inhibitor (cathepsin L inhibitor II, Z-FY-CHO, 10 μM, Calbiochem) as well as two cathepsin B inhibitors: CA-074 (cell unpermeant) and CA-074Me (cell permeant) (both given at a concentration of 10 μM, Calbiochem, Nottingham, UK).

    Techniques: In Vitro, Positive Control

    Cathepsin B expression in cells from primary and metastatic human melanomas . Western blot analysis (left panels) of cathepsin B, cathepsin D, cathepsin L and tubulin in the primary melanoma cell lines PM1 ( A ) and PM2 ( B) and in the metastatic melanoma cell lines MM1 ( A ) and MM2 ( B ). Quantitative flow cytometry analysis (right panels) of plasma membrane cathepsin B in PM1 ( A ) and PM2 ( B ) cell lines and in MM1 ( A ) and MM2 ( B ) cell lines. The values of Western blot signals, reported in the bottom panels of ( A ) and ( B ), were obtained by densitometric analysis and expressed as arbitrary units (a.u.). Numbers in the right panels of ( A ) and ( B ) represent the median fluorescence values. In the right panels of ( A ) and ( B ) statistical analysis performed by non-parametric K/S test is reported.

    Journal: Molecular Cancer

    Article Title: Cathepsin B inhibition interferes with metastatic potential of human melanoma: an in vitro and in vivo study

    doi: 10.1186/1476-4598-9-207

    Figure Lengend Snippet: Cathepsin B expression in cells from primary and metastatic human melanomas . Western blot analysis (left panels) of cathepsin B, cathepsin D, cathepsin L and tubulin in the primary melanoma cell lines PM1 ( A ) and PM2 ( B) and in the metastatic melanoma cell lines MM1 ( A ) and MM2 ( B ). Quantitative flow cytometry analysis (right panels) of plasma membrane cathepsin B in PM1 ( A ) and PM2 ( B ) cell lines and in MM1 ( A ) and MM2 ( B ) cell lines. The values of Western blot signals, reported in the bottom panels of ( A ) and ( B ), were obtained by densitometric analysis and expressed as arbitrary units (a.u.). Numbers in the right panels of ( A ) and ( B ) represent the median fluorescence values. In the right panels of ( A ) and ( B ) statistical analysis performed by non-parametric K/S test is reported.

    Article Snippet: For cell migration and invasion assays, cells were seeded in the presence of the cathepsin D inhibitor (Pepstatin A, 100 μM, Calbiochem), the cathepsin L inhibitor (cathepsin L inhibitor II, Z-FY-CHO, 10 μM, Calbiochem) as well as two cathepsin B inhibitors: CA-074 (cell unpermeant) and CA-074Me (cell permeant) (both given at a concentration of 10 μM, Calbiochem, Nottingham, UK).

    Techniques: Expressing, Western Blot, Flow Cytometry, Cytometry, Fluorescence

    Effect of cathepsin L inhibitor on the bFGF -CM-induced HUVECs migration. (A) HUVECs were incubated with bFGF -CM alone or medium containing MMP-1 neutralizing antibody (5 µg/ml), PAI-1 neutralizing antibody (5 µg/ml), or cathepsin L inhibitor (200 nM) for 12 h. Migrated cells were stained and quantified. The results represent the means ± SEM of three different experiments. 1, LacZ-CM; 2, bFGF-CM; 3, bFGF-CM + MMP-1 neutralizing Ab (5 µg/ml); 4, bFGF-CM + PAI-1 neutralizing Ab (5 µg/ml); 5, bFGF-CM + Cathepsin L inhibitor (200 nM). * P

    Journal: Experimental & Molecular Medicine

    Article Title: Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

    doi: 10.3858/emm.2011.43.4.022

    Figure Lengend Snippet: Effect of cathepsin L inhibitor on the bFGF -CM-induced HUVECs migration. (A) HUVECs were incubated with bFGF -CM alone or medium containing MMP-1 neutralizing antibody (5 µg/ml), PAI-1 neutralizing antibody (5 µg/ml), or cathepsin L inhibitor (200 nM) for 12 h. Migrated cells were stained and quantified. The results represent the means ± SEM of three different experiments. 1, LacZ-CM; 2, bFGF-CM; 3, bFGF-CM + MMP-1 neutralizing Ab (5 µg/ml); 4, bFGF-CM + PAI-1 neutralizing Ab (5 µg/ml); 5, bFGF-CM + Cathepsin L inhibitor (200 nM). * P

    Article Snippet: Neutralizing antibodies against MMP-1 and PAI-1, and cell-impermeable cathepsin L inhibitor (cathepsin inhibitor II, Z-Phe-Tyr-CHO) were also purchased from Calbiochem.

    Techniques: Migration, Incubation, Staining

    Expression and secretion of MMP-1, PAI-1 and cathepsin L by bFGF protein treatment in SkMCs. (A) SkMCs were treated with bFGF protein (1 or 10 ng/ml). After 24 h, total RNA was isolated from SkMCs and RT-PCR was performed. The PCR products were electrophoresed on 1.5% agarose gel. Bands were quantified by densitometry, and the values were normalized to that of β-actin mRNA. * P

    Journal: Experimental & Molecular Medicine

    Article Title: Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

    doi: 10.3858/emm.2011.43.4.022

    Figure Lengend Snippet: Expression and secretion of MMP-1, PAI-1 and cathepsin L by bFGF protein treatment in SkMCs. (A) SkMCs were treated with bFGF protein (1 or 10 ng/ml). After 24 h, total RNA was isolated from SkMCs and RT-PCR was performed. The PCR products were electrophoresed on 1.5% agarose gel. Bands were quantified by densitometry, and the values were normalized to that of β-actin mRNA. * P

    Article Snippet: Neutralizing antibodies against MMP-1 and PAI-1, and cell-impermeable cathepsin L inhibitor (cathepsin inhibitor II, Z-Phe-Tyr-CHO) were also purchased from Calbiochem.

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Effect of cathepsin L on HUVEC migration. (A) HUVEC migration was stimulated by addition of MMP-1, PAI-1 or cathepsin L. After 12 h, migrated cells were stained and quantified. The results represent the means ± SEM of three different experiments. * P

    Journal: Experimental & Molecular Medicine

    Article Title: Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

    doi: 10.3858/emm.2011.43.4.022

    Figure Lengend Snippet: Effect of cathepsin L on HUVEC migration. (A) HUVEC migration was stimulated by addition of MMP-1, PAI-1 or cathepsin L. After 12 h, migrated cells were stained and quantified. The results represent the means ± SEM of three different experiments. * P

    Article Snippet: Neutralizing antibodies against MMP-1 and PAI-1, and cell-impermeable cathepsin L inhibitor (cathepsin inhibitor II, Z-Phe-Tyr-CHO) were also purchased from Calbiochem.

    Techniques: Migration, Staining

    Altered IR-induced nuclear translocation of NF-κB following selective inhibition of cathepsin L with Z-FY-CHO or cathepsin L shRNA. (A) p65 protein immunofluorescence in U251 cells treated with Z-FY-CHO, IR, or both. (B) p65 protein immunofluorescence in in U251-consh and U251-Lsh cells treated with IR (10 Gy).

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of cathepsin L sensitizes human glioma cells to ionizing radiation in vitro through NF-κB signaling pathway

    doi: 10.1038/aps.2014.148

    Figure Lengend Snippet: Altered IR-induced nuclear translocation of NF-κB following selective inhibition of cathepsin L with Z-FY-CHO or cathepsin L shRNA. (A) p65 protein immunofluorescence in U251 cells treated with Z-FY-CHO, IR, or both. (B) p65 protein immunofluorescence in in U251-consh and U251-Lsh cells treated with IR (10 Gy).

    Article Snippet: Antibodies and reagents Cathepsin L inhibitor II (Z-FY-CHO) was purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Translocation Assay, Inhibition, shRNA, Immunofluorescence

    Reduced NF-κB-dependent transcriptional activation in U251 cells following IR with selective inhibition of catheps in L with Z-FY-CHO (A) or cathepsin L shRNA (B). NF-κB activity was assessed using an NF-κB-RE firefly luciferase reporter vector (Promega). For normalization, cells were cotransfected with a constitutive Renilla luciferase reporter (Promega). b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of cathepsin L sensitizes human glioma cells to ionizing radiation in vitro through NF-κB signaling pathway

    doi: 10.1038/aps.2014.148

    Figure Lengend Snippet: Reduced NF-κB-dependent transcriptional activation in U251 cells following IR with selective inhibition of catheps in L with Z-FY-CHO (A) or cathepsin L shRNA (B). NF-κB activity was assessed using an NF-κB-RE firefly luciferase reporter vector (Promega). For normalization, cells were cotransfected with a constitutive Renilla luciferase reporter (Promega). b P

    Article Snippet: Antibodies and reagents Cathepsin L inhibitor II (Z-FY-CHO) was purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Activation Assay, Inhibition, shRNA, Activity Assay, Luciferase, Plasmid Preparation

    The inhibition of cathepsin L sensitized U251 cells to IR. (A) Clonogenic survival curves for U251 cells treated with radiation (0, 2, 4, 6, and 8 Gy) alone or in combination with Z-FY-CHO. (B) Western blot of CTSL in whole cell extracts from parental U251, U251-consh and U251-Lsh cells. β-Actin was used as an internal control. c P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of cathepsin L sensitizes human glioma cells to ionizing radiation in vitro through NF-κB signaling pathway

    doi: 10.1038/aps.2014.148

    Figure Lengend Snippet: The inhibition of cathepsin L sensitized U251 cells to IR. (A) Clonogenic survival curves for U251 cells treated with radiation (0, 2, 4, 6, and 8 Gy) alone or in combination with Z-FY-CHO. (B) Western blot of CTSL in whole cell extracts from parental U251, U251-consh and U251-Lsh cells. β-Actin was used as an internal control. c P

    Article Snippet: Antibodies and reagents Cathepsin L inhibitor II (Z-FY-CHO) was purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Inhibition, Western Blot

    Altered IR-induced nuclear translocation of NF-κB following selective inhibition of cathepsin L with Z-FY-CHO or cathepsin L shRNA. (A) Western blot analysis of IκBα expression and the nuclear translocation of NF-κB in U251 cells treated with Z-FY-CHO, IR, or both. Lamin B was used as a nuclear internal control. c P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of cathepsin L sensitizes human glioma cells to ionizing radiation in vitro through NF-κB signaling pathway

    doi: 10.1038/aps.2014.148

    Figure Lengend Snippet: Altered IR-induced nuclear translocation of NF-κB following selective inhibition of cathepsin L with Z-FY-CHO or cathepsin L shRNA. (A) Western blot analysis of IκBα expression and the nuclear translocation of NF-κB in U251 cells treated with Z-FY-CHO, IR, or both. Lamin B was used as a nuclear internal control. c P

    Article Snippet: Antibodies and reagents Cathepsin L inhibitor II (Z-FY-CHO) was purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Translocation Assay, Inhibition, shRNA, Western Blot, Expressing

    The expression of cathepsin L protein and nuclear translocation of NF-κB p65 and p50 in U251 cells following IR was analyzed by Western blot. (A) Western blot analysis of cathepsin L in U251 cells treated with IR (10 Gy). β-Actin was used as an internal control. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of cathepsin L sensitizes human glioma cells to ionizing radiation in vitro through NF-κB signaling pathway

    doi: 10.1038/aps.2014.148

    Figure Lengend Snippet: The expression of cathepsin L protein and nuclear translocation of NF-κB p65 and p50 in U251 cells following IR was analyzed by Western blot. (A) Western blot analysis of cathepsin L in U251 cells treated with IR (10 Gy). β-Actin was used as an internal control. b P

    Article Snippet: Antibodies and reagents Cathepsin L inhibitor II (Z-FY-CHO) was purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: Expressing, Translocation Assay, Western Blot

    Effect of p65 shRNA on the expression of p65 and cathepsin L in U251 and MCF-7 cells. (A) Western blot of p65 whole cell extracts from normal U251, U251-consh and U251-p65sh cells. β-Actin was used as an internal control. c P

    Journal: Acta Pharmacologica Sinica

    Article Title: Inhibition of cathepsin L sensitizes human glioma cells to ionizing radiation in vitro through NF-κB signaling pathway

    doi: 10.1038/aps.2014.148

    Figure Lengend Snippet: Effect of p65 shRNA on the expression of p65 and cathepsin L in U251 and MCF-7 cells. (A) Western blot of p65 whole cell extracts from normal U251, U251-consh and U251-p65sh cells. β-Actin was used as an internal control. c P

    Article Snippet: Antibodies and reagents Cathepsin L inhibitor II (Z-FY-CHO) was purchased from Calbiochem (La Jolla, CA, USA).

    Techniques: shRNA, Expressing, Western Blot