rat monoclonal antibody against mouse lamp2  (Developmental Studies Hybridoma Bank)

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Lysosomal accumulations of mTOR are lost in concanamycin A (ConA)-treated cells (100 nM, 6 h). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of mTOR/LAMP2 colocalization in (b). n = 50 individual cells from 5 independent fields per condition. (c) ConA treatment (100 nM) preferentially diminishes phosphorylation of the lysosomal substrate TFEB but not of the cytoplasmic substrates S6K and 4E-BP1 under basal culture conditions. ConA (or DMSO as control, Ctrl) was added directly in the media for 6 hours before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (–AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. ConA (or DMSO) was also included in the treatment media. The composition of all media is described in the Methods (see ‘Cell culture treatments’). (d-f) As in (a-c) but for treatments with chloroquine (CQ; 50 μM, 6 h). Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. For all panels, representative data from one out of three independent replicate experiments are shown. Data in graphs shown as mean ± SEM. **** p < 0.0001. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Phospho-proteomics, Control, Lysis, Cell Culture

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal function by BafA1 targeting the v-ATPase. b , c , Basal lysosomal proteolysis in HEK293FT cells shown by accumulation of LC3B upon BafA1 treatment (100 nM, 6 h before fixation) ( b ) and quantification of LC3B signal ( c ). n Ctrl = 49 and n BafA1 = 50 individual cells from five independent fields per condition. d , A schematic representation of the treatment strategy followed in this study, assessing mTORC1 activity under basal (unchallenged cells), starvation or acute re-activation (AA add-back) conditions. AA levels are shown by a black line, and mTORC1 activity by a red line (see also ). e , f , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, treated as indicated, using confocal microscopy (magnified insets shown on the right; scale bars, 25 μm and for insets, 5 μm) ( e ) and quantification of colocalization ( f ). n = 50 individual cells from five independent fields per condition. g , Immunoblots with lysates from HEK293FT WT cells treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and BafA1 as shown, probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. For e – g , BafA1 (100 nM) (or DMSO as control, Ctrl) was added directly in the media for 6 h before fixation ( e and f ) or lysis ( g ). For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation or lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. BafA1 (or DMSO) was also included in the treatment media. The composition of all media is described in . Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. and .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Inhibition, Activity Assay, Activation Assay, Marker, Confocal Microscopy, Western Blot, Control, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal proteases by PepA and E64 blocking local AA production. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells ( b ) and its quantification ( c ), treated as indicated, using confocal microscopy. PepA (50 μM) and E64 (25 μM) (or DMSO as control, Ctrl) were added directly in the media for 16 h before fixation (magnified insets shown to the right; scale bars, 25 μm and for insets, 5 μm). n = 56 individual cells from three independent fields per condition. Data shown as mean ± s.e.m. **** P < 0.0001. d , Immunoblots with lysates from HEK293FT WT cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and protease inhibitors (PepA + E64) as shown, probed with the indicated antibodies. PepA (50 μM) and E64 (25 μM) were added directly in the media for 16 h before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. PepA + E64 (or DMSO) were also included in the treatment media. The composition of all media is described in . Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Inhibition, Blocking Assay, Marker, Confocal Microscopy, Control, Western Blot, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of lysosomal enzyme sorting at the Golgi and delivery to lysosomes that depends on the GNPTAB enzyme. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells using confocal microscopy ( b ) and its quantification ( c ). Cells were transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. The composition of all media is described in . n = 44–50 individual cells from five independent fields per condition (see also ). d , Immunoblots with lysates from HEK293FT WT cells transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions as described in b , probed with the indicated antibodies. e , f , Functional characterization of GNPTAB KO HEK293FT cells. g , h , Lysosomal accumulations of mTOR are lost in GNPTAB KOs ( g ) and quantification of mTOR/LAMP2 colocalization ( h ). n = 50 individual cells from five independent fields per condition. For microscopy, magnified insets are shown to the right. Scale bars, 25 μm and for insets, 5 μm. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Transfection, Control, Western Blot, Functional Assay, Microscopy

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , b , mTOR/LAMP2 colocalization ( a ) and its quantification ( b ). mTOR delocalizes away from lysosomes already after 2 h of BafA1 treatment. Time course of BafA1 treatment (100 nM, 2–8 h) to block lysosomal function in HEK293FT cells. Magnified insets shown to the right ( a ). Scale bars, 25 μm and insets, 5 μm. n = 49–50 individual cells from five independent fields per condition (see also ). c – e , Dephosphorylation kinetics of lysosomal (TFEB) and cytoplasmic (S6K and 4E-BP1) substrates of mTORC1 upon BafA1 treatment (100 nM, 1–8 h) in HEK293FT cells showing a rapid drop in TFEB phosphorylation, whereas that of S6K/4E-BP1 remains largely unaffected even at much later timepoints ( c ). Quantification of TFEB phosphorylation in ( d ) and S6K phosphorylation in ( e ). f , g , The rapamycin time course (20 nM, 1–30 min) in control (WT) and RagA/B KO cells, assessing S6K dephosphorylation kinetics ( f ) and the quantification of S6K phosphorylation ( g ). The rate of S6K dephosphorylation is similar between Rag-proficient and Rag-deficient cells. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Blocking Assay, De-Phosphorylation Assay, Phospho-proteomics, Control

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model for the genetic removal of the Rag GTPases. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT and RagA/B KO cells ( b ) and its quantification ( c ), when treated as indicated, using confocal microscopy. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . Magnified insets shown to the right in b . Scale bars, 25 μm and for insets, 5 μm. n = 55–60 individual cells from three or four independent fields per condition (see also ). d , Lyso-IP experiments with WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes were immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in lysosomal and non-lysosomal fractions as well as in whole-cell lysates was analysed by immunoblotting. e , Immuno-EM analysis of mTOR localization. Control (WT) or RagA/B KO MEFs, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, treated as described in b , were stained with antibodies against endogenous mTOR (10 nm gold particles) and LAMP2 (5 nm gold particles) ( e ). Magnified insets shown on the right side; the area used for magnification is marked with a white square. Scale bars, 500 nm and for insets, 80 nm. LY, LAMP2-positive lysosomes. f , g , Quantification of mTOR localization at lysosomes or the cytoplasm in WT ( f ) or RagA/B KO MEFs ( g ), treated and analysed by immuno-EM as in e . Samples incubated with secondary antibodies only (no primary ab) were used as negative controls for background staining. Values represent number of gold particles per μm 2 . n WT = 58–60 ( f ), n KO = 60 ( g ) randomly selected areas (1 μm 2 each) from three independent grids per condition. Data shown as mean ± s.e.m. * P < 0.05, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Fig. .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Stable Transfection, Expressing, Negative Control, Western Blot, Control, Staining, Incubation

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT or RagC/D KO cells, treated as indicated in the figure, using confocal microscopy. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of colocalization in (b). n WT(+AA) = 50, n CDKO(+AA) = 50, n WT(–AA) = 48, n CDKO(–AA) = 49, n WT(–/+AA) = 49, n CDKO(–/+AA) = 50 individual cells from 5 independent fields per condition. Representative data from one out of three independent experiments are shown. (c) Immunoblots with lysates from HEK293FT WT and RagC/D KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. Treatments were performed as in (a). n = 3 independent experiments. (d-e) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in WT or RagA/B KO MEF cells, treated as indicated in the figure, using confocal microscopy. Treatments were performed as in (a). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (d). Quantification of colocalization in (e). n WT(+AA) = 51, n ABKO(+AA) = 50, n WT(–AA) = 49, n ABKO(–AA) = 50, n WT(–/+AA) = 49, n ABKO(–/+AA) = 49 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown. (f) As in (c), but with WT and RagA/B KO MEFs. n = 3 independent experiments. (g) As in (c), but with WT and RagA/B KO SW-620 cells. n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form; S: SUMOylated form. Data in graphs shown as mean ± SEM. *** p < 0.001, **** p < 0.0001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , Immunoblots with lysates from HEK293FT WT and RagA/B KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . b , In vitro kinase assays with mTORC1 immunopurified from WT or RagA/B KO HEK293FT cells and recombinant 4E-BP1 protein used as substrate, with 4E-BP1 phosphorylation detected by immunoblotting. No ATP samples (−ATP) used as negative controls. c , Lyso-IP experiments in WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in the lysosomal and non-lysosomal fractions, as well as in whole-cell lysates, analysed by immunoblotting. Note the absence of S6K from lysosomal fractions and the presence of phospho-TFEB in the lysosomal fractions only of control cells. n = 2 independent experiments. d , e , Phosphorylation of multiple mTORC1 substrates is largely unaffected by BafA1 treatment (100 nM, 6 h) ( d ) or loss of Rag GTPases ( e ). In e , Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. f , g , GRASP55 phosphorylation by mTORC1 is retained in RagA/B KO ( f ) or BafA1-treated cells (100 nM, 6 h) ( g ), similarly to that of S6K. In g , starvation was performed as in a . Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. h , RagC is an additional lysosomal mTORC1 substrate that requires properly functioning lysosomes for its phosphorylation, similarly to TFEB/TFE3. AA starvation or blockage of lysosomal function with BafA1 (100 nM, 6 h) decrease RagC phosphorylation (shown as elevated RagC signal with #5466). Treatments performed as in a . i , j , Lysosomal localization of RagC is unaffected by BafA1 treatment (100 nM, 6 h) ( i ). Quantification of RagC/LAMP2 colocalization in ( j ). Scale bars, 25 μm and for insets, 5 μm. n = 50 individual cells from five independent fields per condition. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. – .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Western Blot, Lysis, In Vitro, Recombinant, Phospho-proteomics, Stable Transfection, Expressing, Negative Control, Control, Inhibition

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Schematic model of cytoplasmic AA sensing and signaling upstream of the Rags. See text for details. (b-c) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (b). Quantification of colocalization in (c). n siCtrl(+AA) = 49, n siMios(+AA) = 46, n siCtrl(–AA) = 49, n siMios(–AA) = 47, n siCtrl(–/+AA) = 50, n siMios(–/+AA) = 46 individual cells from 5 independent fields per condition. Representative data from one out of two independent experiments are shown. (d) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl), and treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA; 10 or 30 min) conditions, probed with the indicated antibodies. Treatments were performed as in (b). n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. Data in (c) shown as mean ± SEM. * p < 0.05, *** p < 0.001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Transfection, Control, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Expression analysis of LAMTOR1 by qPCR confirms successful knockdown in HEK293FT cells. n = 2 independent experiments. (b) Schematic model of lysosomal tethering of the Rag dimer by the LAMTOR complex. (c-d) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl). Magnified insets shown to the right. Scale bars = 10 μm (for insets, 5 μm) (c). Quantification of colocalization in (d). n siCtrl = 50, n siLAMTOR1 = 48 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown as mean ± SEM. **** p < 0.001. (e) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl), cultured under basal conditions, and probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. n = 3 independent experiments. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Expressing, Knockdown, Marker, Confocal Microscopy, Transfection, Control, Western Blot, Cell Culture

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: Induction of lysosomal membrane permeabilization/rupture in hepatocytes by HNE. A , Changes in lysosomes of HepG2 cells after the addition of EPI and HNE were observed by time-lapse imaging using LysoTracker. Blue , Hoechst; red , LysoTracker. Square on the top right indicates morphological changes that were observed on bright field imaging. B , Electron microscopy images of HepG2 cells. Black squares show magnified images of membrane-bound lysosomes in the control (Cont) and disruption of the lysosomal limiting membrane in HNE-treated HepG2 cells (HNE), respectively. L, lysosome. C , HepG2 cells were treated with 25 μM HNE, and imaged 0, 2, and 6 hours later. Blue , DAPI; green , CTSB; red , LysoTracker; yellow , merge. D , Area occupied by LysoTracker and CTSB for each particle in HepG2 cells at a 20× image. The analysis was performed with 10 images in each group. E , Images show immunoreactivity of HepG2 cells for CTSB and LAMP2 before (0) and 6 hours after the treatment of 25 μM HNE. Blue , DAPI; green , CTSB; red , LAMP2; yellow , merge. F , Electron microscopy images of Huh-7. Black squares show magnified images of membrane-bound lysosomes in the control (Cont) and disruption of the lysosomal limiting membrane in HNE-treated Huh7 cells (HNE), respectively. L, lysosome. G , Huh-7 cells were treated with 25 μM HNE, and imaged 0, 2, and 6 hours later. Blue , DAPI; green , CTSB; red , LysoTracker; yellow , merge. H , Area occupied by LysoTracker and CTSB for each particle in Huh7 cells at a 20× image of Huh7. I , Images show immunoreactivity of Huh-7 cells for CTSB and LAMP2 before (0) and 6 hours after the treatment of 25 μM HNE. Blue , DAPI; green , CTSB; red , LAMP2; yellow , merge.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK) at a dilution of 250, a rabbit monoclonal antibody against human cathepsin B (CTSB) (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 800, a rat monoclonal antibody against mouse LAMP2 (6A430, Santa Cruz Biotechnology, Dallas, TX) at a dilution of 500, and a rabbit monoclonal antibody against mouse CTSB (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 1000.

Techniques: Imaging, Electron Microscopy

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: Lysosomal membrane permeabilization/rupture in hepatocytes of patients with NASH. A , Immunofluorescence staining of liver tissue from patients with nonfatty liver disease and NASH. Blue , DAPI; green , CTSB; red , LAMP2. The area highlighted in the yellow square is a magnified image of the nonfatty liver, whereas the red square is a magnified image of the NASH liver. B , Relationship between the HNE staining score (patient numbers; grade 0, n = 5; grade 1, n = 9; grade 2, n = 12) and granule sizes for LAMP2. The area of particles stained with LAMP2 in 20× image was calculated, respectively. C , Electron microscopy images of the non-fatty liver and NASH liver. Lysosomes with clear limiting membrane structures were observed in the nonfatty liver ( white arrowheads ). In contrast, lysosomes in the NASH liver showed disintegrity ( yellow arrowheads ). Furthermore, in some lysosomes, the lysosomal membrane was disrupted, and contents leaked out ( yellow arrow ). D , A Western blotting analysis of μ-calpain in the livers of 5 patients with nonfatty liver (normal) and 5 patients with NASH is shown. P, protein marker. E , Bands of panel D are quantified and shown as relative fold ratios.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK) at a dilution of 250, a rabbit monoclonal antibody against human cathepsin B (CTSB) (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 800, a rat monoclonal antibody against mouse LAMP2 (6A430, Santa Cruz Biotechnology, Dallas, TX) at a dilution of 500, and a rabbit monoclonal antibody against mouse CTSB (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 1000.

Techniques: Immunofluorescence, Staining, Electron Microscopy, Western Blot, Marker

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: HNE induces lysosomal disintegrity by activating μ -calpain in hepatocytes of Japanese macaque monkeys. A , Macroscopic findings of livers in the control (Cont) and in monkeys treated with HNE (HNE). Black arrows show regional discoloration. B , Hematoxylin and eosin staining and HNE immunostaining of liver tissue from the control group (Cont) and HNE-treated group (HNE). HNE immunoreactivity was observed in hepatocytes. C , The expression of liver HNE protein adducts in the control (Cont) and in monkeys treated with HNE (HNE) was evaluated by a Western blotting analysis. In the Western blotting analysis, HNE is depicted as HNE protein adducts of various molecular weights. P, protein marker. D , Bands of panel C are quantified and shown as relative fold ratios. E , Alterations in ALT levels before the HNE treatment and increases after the HNE treatment are shown. F , Comparison of immunofluorescence staining in the control group (Cont) and HNE-treated group (HNE). Blue , DAPI; green , CTSB; red , LAMP2; yellow , merge. The yellow square shows a magnified image of the liver in the control group, whereas the red square shows a magnified image of the liver in the HNE-treated group. G , The area of particles stained with LAMP2 is shown in 20× image for each monkey. H , The number of stained areas that were 10 μm 2 or larger in panel G is shown. I , Electron microscopy images of livers in the control (Cont) and HNE-treated groups (HNE). Black squares show magnified images of membrane-bound lysosomes in the control (Cont) and disruption of the lysosomal limiting membrane in the HNE-treated group (HNE). L, lysosome. J , A Western blotting analysis showing the expression of activated μ-calpain in the livers of the control (Cont) and HNE-treated groups (HNE). P, protein marker. K , Bands of panel J are quantified and shown as relative fold ratios.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK) at a dilution of 250, a rabbit monoclonal antibody against human cathepsin B (CTSB) (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 800, a rat monoclonal antibody against mouse LAMP2 (6A430, Santa Cruz Biotechnology, Dallas, TX) at a dilution of 500, and a rabbit monoclonal antibody against mouse CTSB (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 1000.

Techniques: Staining, Immunostaining, Expressing, Western Blot, Marker, Immunofluorescence, Electron Microscopy

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis

doi: 10.1016/j.jcmgh.2022.06.008

Figure Lengend Snippet: Administration of Alda-1 in CDAA mice suppresses liver fibrosis. A , Hematoxylin and eosin staining and HNE immunostaining of liver tissue from the control mice fed the standard diet (Cont), CDAA mice (CDAA) and Alda-1-treated CDAA mice (CDAA+Alda-1) for 8 weeks. B , The expressions of liver HNE protein adducts in CDAA mice (CDAA) and CDAA mice with Alda-1 treatment (CDAA+Alda-1) for 4 (4w) and 8 (8w) weeks were evaluated by a Western blotting analysis. C , Bands of panel B are quantified and shown as relative fold ratios. D , Comparison of histopathological scores of CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 8 weeks, such as steatosis score, lobular inflammation score (Lobular inf), ballooning score, and NAFLD activity score (NAS) is shown. E , Sirius red staining of liver tissue from CDAA mice (CDAA) and Alda-1-treated CDAA mice (CDAA+Alda-1) for 8 weeks. F , Area occupied by Sirius red staining in a 20× image were compared between the CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 8 weeks. The area was calculated for each of the 10 images and averaged. G , Real-time PCR analysis of IL-1β, IL-6, tumor necrosis factor (TNF), and toll-like receptor (TRL)-4 expression in CDAA mice ( blue circle ) and Alda-1 treated CDAA mice ( red triangle ) for 4 and 8 weeks. H , Immunofluorescence staining of livers in control mice (Cont) and the CDAA mice (CDAA). Blue , DAPI; green , CTSB; red , LAMP2. I , Electron microscopy images of liver tissue collected from control mice (Cont) and CDAA mice (CDAA) for 8 weeks. The black arrow shows the disruption of the lysosomal limiting membrane. L, lysosome. J , Immunofluorescence LAMP2 staining of liver tissue from CDAA mice (CDAA) and Alda-1-treated CDAA mice (CDAA+Alda-1) for 8 weeks. K , The area of particles stained with LAMP2 in CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 4 weeks is shown. L , The number of stained areas that were 10 μm 2 or larger in panel K is shown. M , The area of particles stained with LAMP2 in CDAA mice (CDAA) and Alda-1 treated CDAA mice (CDAA+Alda-1) for 8 weeks is shown. N , The number of stained areas that were 10 μm 2 or larger in panel M is shown.

Article Snippet: The following primary antibodies were used: a mouse monoclonal antibody against African green monkey LAMP2 (ab25631, Abcam, Cambridge, UK) at a dilution of 250, a rabbit monoclonal antibody against human cathepsin B (CTSB) (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 800, a rat monoclonal antibody against mouse LAMP2 (6A430, Santa Cruz Biotechnology, Dallas, TX) at a dilution of 500, and a rabbit monoclonal antibody against mouse CTSB (D1C7Y, Cell Signaling, Danvers, MA) at a dilution of 1000.

Techniques: Staining, Immunostaining, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Electron Microscopy