cathepsin inhibitor ca 074 me  (MedChemExpress)

Journal: PLoS ONE

Article Title: A2E Induces IL-1ß Production in Retinal Pigment Epithelial Cells via the NLRP3 Inflammasome

doi: 10.1371/journal.pone.0067263

Figure Lengend Snippet: Undifferentiated ARPE-19 cells were pre-stimulated for 48 hours with 1000 pg/ml IL-1α. During the last 24 hours of pre-stimulation, 0, 10 or 20 µM of cathepsin-B inhibitor was added. The medium was then exchanged for serum free DMEM containing 10 µM A2E, along with the cathepsin-B inhibitor (at the same concentration as during the preceding pre-stimulation step). After 24 hours the cell culture supernatant was collected and processed using ELISA. Cathepsin-B inhibitor stock was dissolved in DMSO. Therefore DMSO at the same concentration, but without cathepsin-B inhibitor, was used as a negative control. The effect of cathepsin-B inhibition on ATP (20 µM) induced IL-1ß production was also assessed. Eight separate wells were stimulated with each concentration (n = 8). Error bars represent standard deviation. (*) 20 µM of Cathepsin-B inhibitor significantly inhibited IL-1ß production as compared to the DMSO control (p<0.0001, one-way ANOVA).

Article Snippet: Human vitronectin, anti-human IL-1ß antibody, and the caspase-1 inhibitor (Z-WEHD-FMK) were obtained from R&D Ltd. Anti-mouse IL-1ß antibody was obtained from Abcam Ltd. ARPE19 cells (passage 22) were obtained from American Type Culture Collection (ATCC) Ltd. Recombinant human IL-1α was obtained from Peprotech Ltd. Cathepsin-B inhibitor (CA-074 Me) and silica gel 60 were obtained from Merck Millipore Ltd, while the mouse anti-human ASC monoclonal antibody was obtained from Millipore Ltd. Short interfering RNA (siRNA), Alexa Fluor secondary antibodies, and Alexa Fluor 647 dextran were obtained from Invitrogen Ltd.

Techniques: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control, Inhibition, Standard Deviation

Journal: Advanced Science

Article Title: Death Pathways of Cancer Cells Modulated by Surface Molecule Density on Gold Nanorods

doi: 10.1002/advs.202102666

Figure Lengend Snippet: The interactions between GNRs and the organelles and the subsequent effects on the death pathways of tumor cells. A) The screen of possible death pathways induced by GNRs by flow cytometry (FCM). B) The cause of necrosis by GNRs that is evaluated by the combination of a necrosis inhibitor, Necrostatin‐1 and GNRs. C) The apoptosis induced by GNRs that is verified by the combination of a Caspase inhibitor, Z‐VAD‐FMK and GNRs as determined by Annexin V‐FITC/PI Apoptosis Detection Kit. The data represent mean value ± s.d. ( n = 3). D) The subcellular localization of GNRs in the organelles as observed by TEM. L, M, and GNRs indicate lysosome, mitochondria, and gold nanorods, respectively. E) The localization of GNRs in lysosomes of MDA‐MB‐231 cells evaluated by CLSM. F) Lysosomal membrane permeability evaluated by the LAMP‐2 protein content checked by WB. Noted here, the GAPDH in Figures and , was the same as conducted in one experiment. G) Lysosomal membrane damage checked by FCM. H,I) The changes in the lysosomal permeability determined by lysosomal membrane proteins LAMP‐2 and the dispersity of lysosomal enzyme (Cathepsin B and Cathepsin D) as imaged by CLMS. The scale bar represents 20 µm. Noted here, the GAPDH in Figures , and was same. J) The enlarged picture of subcellular organelles (lysosomes) and intracellular enzymes distribution corresponding to the cells in white frame in (H) and (I). The scale bar represents 20 µm. K) The intensity of the florescence reflected the Cathepsin B and Cathepsin D as conversed from (J). Statistical significance is evaluated in panels (B), (C), (G), and (K) using an unpaired student's t ‐test ( ＊＊＊ p < 0.001, ＊＊ p < 0.01, ＊ p < 0.05).

Article Snippet: The following chemicals were purchased from Sigma Aldrich: silver nitrate (AgNO 3 ), sodium borohydride (NaBH 4 ), hydrogen tetrachloroaurat(III) (HAuCl 4 ), L‐ascorbic acid, cetyltrimethy‐ lammonium bromide (CTAB), and Fluorescein isothiocyanate (FITC); Fetal calf serum (FCS) was purchased from Gibco; CCK‐8 Kit was purchased from Dojindo Laboratories; Annexin V‐FITC/PI Apoptosis Detection Kit was purchased from Becton, Dickinson and Company; LysoTracker‐Red was purchased from Invitrogen; TNF‐ α ELISA kit was purchased from R&D SYSTEMS; fluorochrome‐conjugated secondary antibody (Invitrogen); JC‐1 Mitochondrial Membrane Potential Assay Kit was purchased from Cayman Chemicals; The following antibodies were purchased from Cell Signaling Technology: anticathepsin B, anticathepsin D, anticaspase 8, anticaspase 9, anticaspase 3, anti‐RIP1, anticytochrome c, anti‐LAMP‐2; The following inhibitors were purchased from Selleck Chemicals: Necrosis inhibitor necrostatin‐1, caspase inhibitor Z‐VAD‐FMK, cathepsin B inhibitor CA‐074‐Me; caspase 8 inhibitor Z‐IETD‐FMK; 18.2 MΩ cm Ultrapure water produced by a Millipore Milli‐Q Plus Water Purification System from Millipore was used in all experiments.

Techniques: Flow Cytometry, Membrane, Permeability

Journal: Advanced Science

Article Title: Death Pathways of Cancer Cells Modulated by Surface Molecule Density on Gold Nanorods

doi: 10.1002/advs.202102666

Figure Lengend Snippet: The effects of GNR's properties on the strength of necrosis and apoptosis. A) The effects of composition of surface of GNRs on the specific toxicity. B) The effects of surface properties of GNRs with distinct L/D ratio on the content of necrosis and apoptosis. Here, the corresponding LMP membrane protein level was reflected in the inserted WB. C) The relation between L / D and necrosis and apoptosis evoked by GNRs with distinct L / D ratio. The data points represent mean ± s. d. ( n = 3). Statistical significance was evaluated using an unpaired student's t ‐test for (B) and (C) ( ＊＊＊ p < 0.001). D) The detail intracellular interaction between the GNRs with distinct L/D ratio and lysosomes further confirmed by TEM and CLSM. Noted here, the scar bar in D is 20 µm. E) The revaluation of molecules caspase 9, caspase 3, and LAMP‐2 related to apoptosis, necrosis, and the key proteins of THEB for necrosis‐to‐apoptosis transition. F) The scheme illustrated the microstructural dimension of GNRs. G) The relation between LD and the normalized surface molecules < ρ surf. molecule > of GNRs with different aseptic ratio, the value of LD was analyzed from GNRs in the TEM.

Article Snippet: The following chemicals were purchased from Sigma Aldrich: silver nitrate (AgNO 3 ), sodium borohydride (NaBH 4 ), hydrogen tetrachloroaurat(III) (HAuCl 4 ), L‐ascorbic acid, cetyltrimethy‐ lammonium bromide (CTAB), and Fluorescein isothiocyanate (FITC); Fetal calf serum (FCS) was purchased from Gibco; CCK‐8 Kit was purchased from Dojindo Laboratories; Annexin V‐FITC/PI Apoptosis Detection Kit was purchased from Becton, Dickinson and Company; LysoTracker‐Red was purchased from Invitrogen; TNF‐ α ELISA kit was purchased from R&D SYSTEMS; fluorochrome‐conjugated secondary antibody (Invitrogen); JC‐1 Mitochondrial Membrane Potential Assay Kit was purchased from Cayman Chemicals; The following antibodies were purchased from Cell Signaling Technology: anticathepsin B, anticathepsin D, anticaspase 8, anticaspase 9, anticaspase 3, anti‐RIP1, anticytochrome c, anti‐LAMP‐2; The following inhibitors were purchased from Selleck Chemicals: Necrosis inhibitor necrostatin‐1, caspase inhibitor Z‐VAD‐FMK, cathepsin B inhibitor CA‐074‐Me; caspase 8 inhibitor Z‐IETD‐FMK; 18.2 MΩ cm Ultrapure water produced by a Millipore Milli‐Q Plus Water Purification System from Millipore was used in all experiments.

Techniques: Membrane

Journal: Advanced Science

Article Title: Death Pathways of Cancer Cells Modulated by Surface Molecule Density on Gold Nanorods

doi: 10.1002/advs.202102666

Figure Lengend Snippet: Necrosis and apoptosis caused by GNRs. A) The confirmation of necrosis and apoptosis according to immunofluorescence (IF) imaging. The apoptosis is evaluated by Caspase 8 inhibitor Z‐IETD‐FMK and Cathepsin B inhibitor CA‐074‐Me by IF. B) The molecular expression of key protein including the cleaved Caspase 8, RIPI, and Cathepsin B as tested by WB. The detailed apoptosis pathway of MDA‐MB‐231 cells detected by C) mitochondrial membrane potential according to JC‐1 assay and D) the leakage of cytochrome c induced by GNRs. E) The expression and activation of Cathepsin D released from lysosomes in tumor cells tested by WB. F) Confirmation of apoptosis based on the protein level of cleaved caspase 9, and cleaved caspase 3 tested by WB. G) The time dependence of apoptosis and necrosis checked by the Annexin V‐FITC/PI Apoptosis Detection Kit by flow cytometry for same GNRs and tumor cells. The data represent mean ± s.d. ( n = 3). H) TNF‐ α level as determined by ELISA. The data represent mean ± s.d. ( n = 3). Statistical significance is evaluated using an unpaired student's t ‐test ( ＊＊＊ p < 0.001). Noted here, the GAPDH in Figure , and Figure was same as conducted in one experiment; the scale bar in A, C, and D is 20 µm.

Article Snippet: The following chemicals were purchased from Sigma Aldrich: silver nitrate (AgNO 3 ), sodium borohydride (NaBH 4 ), hydrogen tetrachloroaurat(III) (HAuCl 4 ), L‐ascorbic acid, cetyltrimethy‐ lammonium bromide (CTAB), and Fluorescein isothiocyanate (FITC); Fetal calf serum (FCS) was purchased from Gibco; CCK‐8 Kit was purchased from Dojindo Laboratories; Annexin V‐FITC/PI Apoptosis Detection Kit was purchased from Becton, Dickinson and Company; LysoTracker‐Red was purchased from Invitrogen; TNF‐ α ELISA kit was purchased from R&D SYSTEMS; fluorochrome‐conjugated secondary antibody (Invitrogen); JC‐1 Mitochondrial Membrane Potential Assay Kit was purchased from Cayman Chemicals; The following antibodies were purchased from Cell Signaling Technology: anticathepsin B, anticathepsin D, anticaspase 8, anticaspase 9, anticaspase 3, anti‐RIP1, anticytochrome c, anti‐LAMP‐2; The following inhibitors were purchased from Selleck Chemicals: Necrosis inhibitor necrostatin‐1, caspase inhibitor Z‐VAD‐FMK, cathepsin B inhibitor CA‐074‐Me; caspase 8 inhibitor Z‐IETD‐FMK; 18.2 MΩ cm Ultrapure water produced by a Millipore Milli‐Q Plus Water Purification System from Millipore was used in all experiments.

Techniques: Immunofluorescence, Imaging, Expressing, Membrane, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Death Pathways of Cancer Cells Modulated by Surface Molecule Density on Gold Nanorods

doi: 10.1002/advs.202102666

Figure Lengend Snippet: The schematic illustration about the relationship between surface molecule density and the cell death pathway. Surface density of CTAB on GNRs largely influences the internalization of GNRs, its intracellular route, the interaction between GNRs and lysosomes, and the specific killing of cancer cells dominated by apoptosis or necrosis. Caspase 8 protease plays a key role in the cell fate transition from necrosis to apoptosis which is tuned by tailoring GNR's Surface molecule density ρ surf. molecule .

Article Snippet: The following chemicals were purchased from Sigma Aldrich: silver nitrate (AgNO 3 ), sodium borohydride (NaBH 4 ), hydrogen tetrachloroaurat(III) (HAuCl 4 ), L‐ascorbic acid, cetyltrimethy‐ lammonium bromide (CTAB), and Fluorescein isothiocyanate (FITC); Fetal calf serum (FCS) was purchased from Gibco; CCK‐8 Kit was purchased from Dojindo Laboratories; Annexin V‐FITC/PI Apoptosis Detection Kit was purchased from Becton, Dickinson and Company; LysoTracker‐Red was purchased from Invitrogen; TNF‐ α ELISA kit was purchased from R&D SYSTEMS; fluorochrome‐conjugated secondary antibody (Invitrogen); JC‐1 Mitochondrial Membrane Potential Assay Kit was purchased from Cayman Chemicals; The following antibodies were purchased from Cell Signaling Technology: anticathepsin B, anticathepsin D, anticaspase 8, anticaspase 9, anticaspase 3, anti‐RIP1, anticytochrome c, anti‐LAMP‐2; The following inhibitors were purchased from Selleck Chemicals: Necrosis inhibitor necrostatin‐1, caspase inhibitor Z‐VAD‐FMK, cathepsin B inhibitor CA‐074‐Me; caspase 8 inhibitor Z‐IETD‐FMK; 18.2 MΩ cm Ultrapure water produced by a Millipore Milli‐Q Plus Water Purification System from Millipore was used in all experiments.

Techniques: