Journal: The Journal of Biological Chemistry

Article Title: Autophagosomal Membrane Serves as Platform for Intracellular Death-inducing Signaling Complex (iDISC)-mediated Caspase-8 Activation and Apoptosis *

doi: 10.1074/jbc.M111.309104

Figure Lengend Snippet: SKI-I simultaneously induces autophagy and caspase-dependent cell death. A, MEFs were treated with 2.5 μm SKI-I for the indicated periods of time and subjected to immunoblot analyses using the indicated antibodies. B, MEFs stably expressing GFP-LC3 were treated with 2.5 μm SKI-I or control DMSO for 12 h and the number of GFP-LC3 dots per cell area (1000 μm2) was determined using a fluorescence microscope (mean ± S.D.; n = 36). Statistical significance was determined by Student's t test. C, MEFs were treated with 2.5 μm SKI-I or control DMSO for 8 h followed by a 4-h co-treatment with 100 nm bafilomycin A1, 25 μm CQ, 20 mm NH4Cl, or control PBS and subjected to immunoblot analyses using the indicated antibodies. D, MEFs were treated with 2.5 μm SKI-I for 12 h, stained with anti-Lamp1 monoclonal and anti-active caspase-3 (C-Casp-3) polyclonal antibodies, and analyzed by fluorescence deconvolution microscopy. Magnified images are shown as insets. E, the fluorescence intensities along the dotted line in D were quantified using SlideBook software. The values of the vertical axis represent fluorescence intensity units (ADU). The horizontal axis represents distance (S, start point; E, end point). F, MEFs were treated with control DMSO or 2.5 μm SKI-I in the presence of 20 μm Z-VAD-fmk or control DMSO for 24 h and cell viability was assessed by measuring cellular ATP levels (mean ± S.D.; n = 3). The scale bars represent 10 μm in A and D, and 1 μm in the insets in D.

Article Snippet: Antibodies were obtained from the following sources: rabbit polyclonal anti-LC3 (Novus Biologicals, NB100-2220 for immunoblot analyses; MBL International, PM046 for immunostaining), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling, 9661), rabbit polyclonal anti-poly(ADP-ribose) polymerase (PARP) (Cell Signaling, 9542), rabbit polyclonal anti-caspase-8 (Cell Signaling, 4927S; R & D Systems, AF1650), mouse monoclonal anti-caspase-8 (Cell Signaling, 9746), rabbit polyclonal anti-Atg16L (MBL International, PM040), mouse monoclonal anti-Atg5 (MBL International, M153-3), mouse monoclonal anti-Bcl-xL (Sigma, B9429), guinea pig polyclonal anti-p62 (American Research Products, Inc., 03-GP62-C), mouse monoclonal anti-FADD (Enzo Life Sciences, AAM-212), rabbit polyclonal anti-DsRed (Clontech, 632496), and mouse monoclonal anti-β-actin (Sigma, A5441).

Techniques: Western Blot, Stable Transfection, Expressing, Fluorescence, Microscopy, Staining, Software

Journal: The Journal of Biological Chemistry

Article Title: Autophagosomal Membrane Serves as Platform for Intracellular Death-inducing Signaling Complex (iDISC)-mediated Caspase-8 Activation and Apoptosis *

doi: 10.1074/jbc.M111.309104

Figure Lengend Snippet: SKI-I promotes caspase-8 self-association in a p62-dependent but Atg5-independent manner. A, Atg5+/+ and Atg5−/− MEFs were transfected with pro-Casp-8 (C360A)-VN and pro-Casp-8 (C360A)-VC for 24 h, treated with 2.5 μm SKI-I for 0 and 6 h, and analyzed by fluorescence microscopy (top) in combination with differential interference contrast microscopy (bottom). B, Atg5+/+ and Atg5−/− MEFs were infected with lentiviruses encoding shScr or shp62 and selected with 1 μg/ml of puromycin. Cells were transfected with pro-Casp-8 (C360A)-VN and pro-Casp-8 (C360A)-VC for 24 h, treated with 2.5 μm SKI-I for 6 h, stained with guinea pig anti-p62 polyclonal antibodies, and analyzed by fluorescence deconvolution microscopy. Arrows indicate colocalization of caspase-8 complexes with p62. The scale bars represent 20 μm in A and 10 μm in B.

Article Snippet: Antibodies were obtained from the following sources: rabbit polyclonal anti-LC3 (Novus Biologicals, NB100-2220 for immunoblot analyses; MBL International, PM046 for immunostaining), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling, 9661), rabbit polyclonal anti-poly(ADP-ribose) polymerase (PARP) (Cell Signaling, 9542), rabbit polyclonal anti-caspase-8 (Cell Signaling, 4927S; R & D Systems, AF1650), mouse monoclonal anti-caspase-8 (Cell Signaling, 9746), rabbit polyclonal anti-Atg16L (MBL International, PM040), mouse monoclonal anti-Atg5 (MBL International, M153-3), mouse monoclonal anti-Bcl-xL (Sigma, B9429), guinea pig polyclonal anti-p62 (American Research Products, Inc., 03-GP62-C), mouse monoclonal anti-FADD (Enzo Life Sciences, AAM-212), rabbit polyclonal anti-DsRed (Clontech, 632496), and mouse monoclonal anti-β-actin (Sigma, A5441).

Techniques: Transfection, Fluorescence, Microscopy, Infection, Staining

Journal: The Journal of Biological Chemistry

Article Title: Autophagosomal Membrane Serves as Platform for Intracellular Death-inducing Signaling Complex (iDISC)-mediated Caspase-8 Activation and Apoptosis *

doi: 10.1074/jbc.M111.309104

Figure Lengend Snippet: SKI-I induces translocation of caspase-8 and FADD to Atg5-positive autophagosomal membranes. A, Atg5−/− MEFs were transfected in combination with Atg5-VN and FADD-VC or control empty VN and VC for 18 h. Cells were fixed, immunostained with anti-LC3 and anti-Atg16L antibodies, and analyzed by fluorescence microcopy. B, Atg5−/− MEFs were infected with retroviruses encoding GFP-Atg5 and selected with 1 μg/ml of puromycin for 5 days. The resultant stable transfectants were treated with 2.5 μm SKI-I for the indicated times and subjected to immunoprecipitation with anti-GFP monoclonal antibodies or control mouse IgG followed by immunoblot analyses using the indicated antibodies. C, Atg5−/− MEFs stably expressing GFP-Atg5 were treated with 2.5 μm SKI-I or control DMSO for 12 h, immunostained with anti-FADD and anti-caspase-8 antibodies, and analyzed by fluorescence deconvolution microcopy. Arrowheads and arrows indicate colocalization of Atg5 with FADD, and Atg5 and FADD-positive signals with caspase-8, respectively. D, the intensity profiles for each fluorescence along the dotted line in C are shown. The scale bars represent 10 μm in A and C, and 5 μm in the inset in C. E, FADD+/+ and FADD−/− MEFs were treated with 5 μm SKI-I or control DMSO for 24 h. Representative micrographs are shown. F, Atg5+/+ MEFs were infected with lentiviruses encoding mStr-Atg4B(C74A) or empty control. After selection with 1 μg/ml of puromycin, the resultant stable transfectants were subjected to treatment with 2.5 μm SKI-I or control DMSO for 16 h. Total cell lysates were subjected to immunoblot analyses using the indicated antibodies. Asterisks indicate nonspecific bands. Cleaved caspase-8 was quantified as the percentage of total caspase-8. Cleaved caspase-3 was quantified as the relative expression after normalization to β-actin.

Article Snippet: Antibodies were obtained from the following sources: rabbit polyclonal anti-LC3 (Novus Biologicals, NB100-2220 for immunoblot analyses; MBL International, PM046 for immunostaining), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling, 9661), rabbit polyclonal anti-poly(ADP-ribose) polymerase (PARP) (Cell Signaling, 9542), rabbit polyclonal anti-caspase-8 (Cell Signaling, 4927S; R & D Systems, AF1650), mouse monoclonal anti-caspase-8 (Cell Signaling, 9746), rabbit polyclonal anti-Atg16L (MBL International, PM040), mouse monoclonal anti-Atg5 (MBL International, M153-3), mouse monoclonal anti-Bcl-xL (Sigma, B9429), guinea pig polyclonal anti-p62 (American Research Products, Inc., 03-GP62-C), mouse monoclonal anti-FADD (Enzo Life Sciences, AAM-212), rabbit polyclonal anti-DsRed (Clontech, 632496), and mouse monoclonal anti-β-actin (Sigma, A5441).

Techniques: Translocation Assay, Transfection, Fluorescence, Infection, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Selection

Journal: Age

Article Title: Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

doi: 10.1007/s11357-011-9234-4

Figure Lengend Snippet: Brain sections of aged mice ( a – i ) are depicted in comparison with those of young mice ( j – l ). In the hippocampal formation of aged mice, there are scattered clusters of puncta ( arrows , higher magnification inset ) immunoreactive for microtubule-associated protein 1 light chain 3 ( LC3 , a ), p62 ( b ), ubiquitin ( Ubi , c ), microtubule-associated protein 2 ( MAP2 , e ), and synapsin IIa ( Syn , f ). These immunoreactive puncta ( arrows , higher magnification inset ) are also present in the olfactory bulb ( d ) and cerebellar cortex ( g ). No clusters of immunoreactive puncta are observed on any of the hemi-brain sections immunostained for phosphorylated Tau ( p-Tau , h ) or synaptophysin ( Syp , i ). Note that hippocampal formation sections ( a – c , e , f , h , i ) are of one aged mouse, and olfactory bulb ( d ) and cerebellar ( g ) sections are of another aged mouse. By contrast, young mice ( j – l , the hippocampal formation of one young mouse) show no ( k , l ) or sparse ( j [ arrow ], higher magnification inset ) clusters of immunoreactive puncta. SO stratum oriens, SP stratum pyramidale, SR stratum radiatum, SL stratum lacunosum-moleculare, SM stratum moleculare, SG stratum granulosum, PZ pleomorphic zone (hilus)

Article Snippet: To evaluate the cellular distribution of autophagosome-related proteins, we used primary antibodies raised against LC3 (rabbit polyclonal, APG8b, #AP1802a, Abgent, San Diego, CA, USA, 1:50 dilution), p62 (rabbit polyclonal, SQSTM1, #AP2183b, Abgent, 1:25), and ubiquitin (mouse monoclonal, #MAB1510, Millipore, Temecula, CA, USA, 1:4,000).

Techniques:

Journal: Age

Article Title: Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

doi: 10.1007/s11357-011-9234-4

Figure Lengend Snippet: Distributions of clustered immunoreactive puncta in the hippocampal subfields, olfactory bulb/tubercle, and cerebellar cortex

Article Snippet: To evaluate the cellular distribution of autophagosome-related proteins, we used primary antibodies raised against LC3 (rabbit polyclonal, APG8b, #AP1802a, Abgent, San Diego, CA, USA, 1:50 dilution), p62 (rabbit polyclonal, SQSTM1, #AP2183b, Abgent, 1:25), and ubiquitin (mouse monoclonal, #MAB1510, Millipore, Temecula, CA, USA, 1:4,000).

Techniques:

Journal: Age

Article Title: Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

doi: 10.1007/s11357-011-9234-4

Figure Lengend Snippet: Double immunofluorescent staining of the hippocampal formation of aged mice shows within clustered puncta co-localization of microtubule-associated protein 2 ( MAP2 , a , d , h ) with microtubule-associated protein 1 light chain 3 ( LC3 , b , CA1 stratum radiatum), p62 ( e , stratum moleculare), and ubiquitin ( Ubi , g , CA1 stratum lacunosum-moleculare [ upper ] and stratum moleculare [ lower ]). Ubiquitin ( j , m , p ) is also co-localized within clustered puncta with synapsin IIa ( Syn , k [ arrows ], CA1 stratum lacunosum-moleculare), LC3 ( n , CA1 stratum radiatum), and p62 ( q , stratum moleculare). c , f , i , l , o , r Each represents a merged image in their respective row, with DAPI-labeled nuclei ( blue ) in c , f , and i ; bar = 20 μm. Note that a – i were obtained from one aged mouse and j – r from another aged mouse

Article Snippet: To evaluate the cellular distribution of autophagosome-related proteins, we used primary antibodies raised against LC3 (rabbit polyclonal, APG8b, #AP1802a, Abgent, San Diego, CA, USA, 1:50 dilution), p62 (rabbit polyclonal, SQSTM1, #AP2183b, Abgent, 1:25), and ubiquitin (mouse monoclonal, #MAB1510, Millipore, Temecula, CA, USA, 1:4,000).

Techniques: Staining, Labeling

Journal: Age

Article Title: Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

doi: 10.1007/s11357-011-9234-4

Figure Lengend Snippet: In the hippocampal formation of all mice studied (22 young and 18 aged), the semiquantitative burden of clustered puncta immunoreactive for microtubule-associated protein 1 light chain 3 ( LC3 , a ), p62 ( b ), ubiquitin ( c ), microtubule-associated protein 2 ( MAP2 , d ), and synapsin IIa ( e ) each shows a significant linear trend toward greater load in aged mice ( p < 0.0001, Chi-square test for linear trend)

Article Snippet: To evaluate the cellular distribution of autophagosome-related proteins, we used primary antibodies raised against LC3 (rabbit polyclonal, APG8b, #AP1802a, Abgent, San Diego, CA, USA, 1:50 dilution), p62 (rabbit polyclonal, SQSTM1, #AP2183b, Abgent, 1:25), and ubiquitin (mouse monoclonal, #MAB1510, Millipore, Temecula, CA, USA, 1:4,000).

Techniques:

Journal: Age

Article Title: Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

doi: 10.1007/s11357-011-9234-4

Figure Lengend Snippet: The linear correlation coefficient ( ρ ) between the numbers of clusters of immunoreactive puncta is shown for each pair of proteins studied in each of three brain regions (i.e., the hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex) of 18 aged mice (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Spearman’s rank correlation). Note whether there were clustered puncta immunoreactive for ubiquitin and synapsin IIa in the cerebellar cortex was undetermined due to intense immunoreactivity of these two proteins in the granule cell layer. LC3 microtubule-associated protein 1 light chain 3, Ubi ubiquitin, MAP2 microtubule-associated protein 2

Article Snippet: To evaluate the cellular distribution of autophagosome-related proteins, we used primary antibodies raised against LC3 (rabbit polyclonal, APG8b, #AP1802a, Abgent, San Diego, CA, USA, 1:50 dilution), p62 (rabbit polyclonal, SQSTM1, #AP2183b, Abgent, 1:25), and ubiquitin (mouse monoclonal, #MAB1510, Millipore, Temecula, CA, USA, 1:4,000).

Techniques:

Journal: Age

Article Title: Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice

doi: 10.1007/s11357-011-9234-4

Figure Lengend Snippet: Scattergraphs with their trend lines show significant inverse correlations between the discrimination ratio (i.e., the ratio of the time spent exploring the novel object over the total amount of time spent exploring both novel and familiar objects during the retention phase of the single-trial object recognition test) as a measure of the short-term recognition memory performance and the number of clusters of puncta immunoreactive for microtubule-associated protein 1 light chain 3 ( LC3 ) and p62 in the hippocampal formation of 18 aged mice ( ρ = −0.48 and −0.55, p = 0.044 and 0.018, respectively, Spearman’s rank correlation)

Article Snippet: To evaluate the cellular distribution of autophagosome-related proteins, we used primary antibodies raised against LC3 (rabbit polyclonal, APG8b, #AP1802a, Abgent, San Diego, CA, USA, 1:50 dilution), p62 (rabbit polyclonal, SQSTM1, #AP2183b, Abgent, 1:25), and ubiquitin (mouse monoclonal, #MAB1510, Millipore, Temecula, CA, USA, 1:4,000).

Techniques: