anti-p62 Search Results


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  • 99
    Millipore anti p62 sqstm1 antibody
    Anti P62 Sqstm1 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti sqstm1 p62
    BRF1 reduced autophagy in RAW264.7 macrophage. (A) Relative mRNA level of BRF1 was measured by qPCR after infection with Ad-GFP or Ad-BRF1 for 24 h in RAW264.7 macrophages. (B) Determination of BRF1 and autophagy related protein LC3, <t>SQSTM1/p62,</t> ATG7, Beclin1, ATG5- ATG12 protein expression level after infection with Ad-GFP or Ad-BRF1 in RAW264.7 macrophage for 24 h by Western blotting analysis. β-actin expression monitored as controls for similar loading of proteins in each lane. (C–F) Quantitative analysis of endogenous LC3(C),SQSTM1/p62 (D), ATG7 (E), Beclin1 (F) expression were shown in bar graphs. Y axis presented the ratio of protein level compared to β-actin respectively. Experiments were repeated at least three times. (G) Cells were incubated with Ad-BRF1/Ad-GFP and LPS for 24 h in 24-wells dish, followed with 50 nmol/ml of autophagy activator Rapamycine for 3 h. Next, it was stained by LC3 (red) and DAPI (blue) and performed by confocal microscopy (400x).
    Anti Sqstm1 P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti p62
    The effect of BA on autophagic flux in KM3 cells. KM3 cells were treated with BA and LC3-II and <t>P62</t> were applied to analyze the autophagic flux using Western blot analysis. The results showed that BA increased the amount of LC3-II and P62 in a dose-dependent
    Anti P62, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti p62
    Effect of SVIP and starvation on cell viability and autophagy in CCl 4 -treated HepG2 cells. a MTT assay showing the viability of HepG2 cells transfected with SVIP-expressing plasmid or vector control treated with CCl 4 or DMSO for 0–72 h. b Western blots (left panel) showing the expression of SVIP, Beclin1, <t>p62,</t> and LC3B in HepG2 cells transfected with siSVIP or siRNA control treated with CCl 4 or DMSO for 24 h. MTT assays (right panel) showing the viability of HepG2 cells transfected with siSVIP or siRNA control treated with CCl 4 or DMSO for 0–72 h. c Western blots showing the expression of SVIP, Beclin1, p62, and LC3B in HepG2 cells treated with CCl 4 or DMSO (12 h) in the absence and presence of starvation (2 h). d Western blots (left panel) and MTT assays (right panel) showing SVIP, p62, Beclin1, and LC3B expression and cell viability of CCl 4 -treated HepG2 cells transfected with SVIP expressing plasmid or vector in the presence or absence of starvation. Starvation started at 12, 36, and 60 h as indicated by arrows. e Western blots (left panel) and MTT assays (right panel) showing the expression of SVIP, Beclin1, p62, and LC3B in CCl 4 -treated HepG2 cells transfected with siSVIP or siRNA control in the absence and presence of starvation. 72 h after siRNA transfection, cells were subjected to CCl 4 treatment in the absence and presence of starvation (45 min). Starvation started at 12 and 36 h as indicated by arrows. Data are presented as mean ± SD in three independent experiments. LC3-II/LC3-I ratio represents the autophagy flux. * p
    Anti P62, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti p62
    Interfering with <t>p62</t> protein level or function increases cell death induced by mutant huntingtin. (A) GFP-positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h of expression, GFP-positive cells attached to the surface with a normal nuclear appearance (open arrowheads) and were scored as live, whereas rounded, partially detached cells with a condensed or fragmented nuclei (arrows) were scored as dead. More than 200 GFP-positive cells in randomly selected microscope views were scored. Bars, 20 μm. (B) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N-HttQ68–induced cell death in HeLa and SHSY-5Y neuroblastoma cells.
    Anti P62, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 372 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc rabbit anti p62
    RNF216 regulated GN11 cells migration through autophagy. (A) Depletion of RNF216 upregulated autophagy flux in GN11 cells. The protein levels of LC3 and <t>P62</t> were detected with immunoblotting in GN11 cells transfected with siNC and siRNF. ACTIN was used as a loading control. (B,C) Quantification of LC3-II (B) and P62 (C) protein levels in GN11 cells as detected by immunoblotting. Data is shown as the mean ± SEM of three independent experiments, * P
    Rabbit Anti P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p62  (Abcam)
    99
    Abcam p62
    Drugs influence the autophagic flux in LTC cells. CHC and CHC plus metfomin (Met) treatment caused accumulation of <t>p62</t> whereas metformin treatment reduced p62 level (A) . Gemcitabine (Gem) treatment reduced the p62 level (B) .
    P62, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1713 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Progen Biotechnik anti p62
    <t>p62</t> does not allow Tax autophagic degradation at the steady-state. ( a ) HeLa cells stably expressing GFP-LC3 were transfected with Tax-His and analyzed by confocal microscopy after staining with His- (white) and p62- or OPTN-specific (red) antibodies. Representative images are shown. Tax, p62 or OPTN and GFP-LC3 signals were quantified along the segment represented on the merge panel and plotted on the histogram. Scale bar = 10 μm. ( b , c ) HeLa cells transiently expressing Tax-His were treated with lysosomal inhibitors ( b , E64D/Pep.) or starved ( c , HBSS) before lysis, western blot analyses and quantification. Full-length blots are presented in Supplementary Fig. S4 . Blots and graphs show results representative of at least 3 experiments.
    Anti P62, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 93/100, based on 306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Progen Biotechnik guinea pig anti p62
    Inhibition of autophagy with CQ sensitizes mesothelioma cell lines to PI3K/mTOR inhibitors. ( a ) <t>Anti-P62,</t> L-C3BI/II and Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated with 20 μ M CQ, 0.5 μ M GDC-0980 or in combination for 24, 48 and 72 h. ( b ) Light micrographs of SPC212 and Mero-82 treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Scale bar represents 25 μ m. ( c ) Quantification of the percentage of ATP content of SPC212 cells and Mero-82 cell treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Data are presented as means±S.D. from ≥3 independent experiments. Significance was determined by analysis of variance test (*** P
    Guinea Pig Anti P62, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 89/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc sqstm1 p62
    Inhibition of autophagy with CQ sensitizes mesothelioma cell lines to PI3K/mTOR inhibitors. ( a ) <t>Anti-P62,</t> L-C3BI/II and Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated with 20 μ M CQ, 0.5 μ M GDC-0980 or in combination for 24, 48 and 72 h. ( b ) Light micrographs of SPC212 and Mero-82 treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Scale bar represents 25 μ m. ( c ) Quantification of the percentage of ATP content of SPC212 cells and Mero-82 cell treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Data are presented as means±S.D. from ≥3 independent experiments. Significance was determined by analysis of variance test (*** P
    Sqstm1 P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 715 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abnova anti p62
    The NBD of NOD2 interacts with both TRAF6 and UBA domains of <t>p62.</t> A . NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. B . HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). C . Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.
    Anti P62, supplied by Abnova, used in various techniques. Bioz Stars score: 93/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Proteintech rabbit p62 sqstm1 polyclonal
    The NBD of NOD2 interacts with both TRAF6 and UBA domains of <t>p62.</t> A . NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. B . HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). C . Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.
    Rabbit P62 Sqstm1 Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson mouse anti p62
    More B. cepacia vacuoles colocalize with <t>p62</t> in WT macrophages than in ΔF508 macrophages. A , confocal microscopy for WT and ΔF508 macrophages infected with B. cepacia -expressing m-RFP for 0.5 or 1.5 h. p62 stained green , whereas nuclei
    Mouse Anti P62, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam p62 sequestosome 1
    More B. cepacia vacuoles colocalize with <t>p62</t> in WT macrophages than in ΔF508 macrophages. A , confocal microscopy for WT and ΔF508 macrophages infected with B. cepacia -expressing m-RFP for 0.5 or 1.5 h. p62 stained green , whereas nuclei
    P62 Sequestosome 1, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem anti p62
    <t>p62</t> deficiency increases the levels of ubiquitinated proteins after methylmercury (MeHg) treatment. Wild-type and p62-defecient mouse embryonic fibroblasts were seeded in 6-cm dishes for treatments. After treatment with 1 µM MeHg for 0–24 h, whole cell lysates were immunoblotted with anti-ubiquitin and p62 antibodies. GAPDH was used as the loading control.
    Anti P62, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    MBL International anti p62
    Super-resolution microscopy (SR-SIM) revealed that the aggresome consists of <t>p62</t> bodies containing p62 and ubiquitinated proteins. N2a cells stably expressing RFP-fused ubiquitin (R-UB) were treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA for 12 h as indicated. S403-phosphorylated p62 (S403-P; green) in ( A ), Lamp2A (lysosome; green) in ( B ), RFP-UB (red), and total p62 (blue) in ( A,B ) were visualized by super-resolution structured illumination microscopy (SR-SIM) as indicated. Magnified images in square box (10 µm each side) on left panels are shown. Scale bars represent 5 µm in wide images or 2 µm in magnified images as indicated.
    Anti P62, supplied by MBL International, used in various techniques. Bioz Stars score: 93/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    BRF1 reduced autophagy in RAW264.7 macrophage. (A) Relative mRNA level of BRF1 was measured by qPCR after infection with Ad-GFP or Ad-BRF1 for 24 h in RAW264.7 macrophages. (B) Determination of BRF1 and autophagy related protein LC3, SQSTM1/p62, ATG7, Beclin1, ATG5- ATG12 protein expression level after infection with Ad-GFP or Ad-BRF1 in RAW264.7 macrophage for 24 h by Western blotting analysis. β-actin expression monitored as controls for similar loading of proteins in each lane. (C–F) Quantitative analysis of endogenous LC3(C),SQSTM1/p62 (D), ATG7 (E), Beclin1 (F) expression were shown in bar graphs. Y axis presented the ratio of protein level compared to β-actin respectively. Experiments were repeated at least three times. (G) Cells were incubated with Ad-BRF1/Ad-GFP and LPS for 24 h in 24-wells dish, followed with 50 nmol/ml of autophagy activator Rapamycine for 3 h. Next, it was stained by LC3 (red) and DAPI (blue) and performed by confocal microscopy (400x).

    Journal: Genes & Diseases

    Article Title: BRF1 ameliorates LPS-induced inflammation through autophagy crosstalking with MAPK/ERK signaling

    doi: 10.1016/j.gendis.2018.04.004

    Figure Lengend Snippet: BRF1 reduced autophagy in RAW264.7 macrophage. (A) Relative mRNA level of BRF1 was measured by qPCR after infection with Ad-GFP or Ad-BRF1 for 24 h in RAW264.7 macrophages. (B) Determination of BRF1 and autophagy related protein LC3, SQSTM1/p62, ATG7, Beclin1, ATG5- ATG12 protein expression level after infection with Ad-GFP or Ad-BRF1 in RAW264.7 macrophage for 24 h by Western blotting analysis. β-actin expression monitored as controls for similar loading of proteins in each lane. (C–F) Quantitative analysis of endogenous LC3(C),SQSTM1/p62 (D), ATG7 (E), Beclin1 (F) expression were shown in bar graphs. Y axis presented the ratio of protein level compared to β-actin respectively. Experiments were repeated at least three times. (G) Cells were incubated with Ad-BRF1/Ad-GFP and LPS for 24 h in 24-wells dish, followed with 50 nmol/ml of autophagy activator Rapamycine for 3 h. Next, it was stained by LC3 (red) and DAPI (blue) and performed by confocal microscopy (400x).

    Article Snippet: 5% nonfat dry milk was used to reduce it nonspecific binding and primary antibodies was incubated overnight at 4 °C: Anti-SQSTM1 (p62), anti-caspase-12, anti-caspase-3, anti-ATG12, anti-BRF1, anti-ATG7, anti-β-actin, anti-ERK1/2, anti-p-ERK1/2 were bought from CST corresponding secondary antibody was incubated for 2 h at 25 °C .

    Techniques: Real-time Polymerase Chain Reaction, Infection, Expressing, Western Blot, Incubation, Staining, Confocal Microscopy

    BRF1 inhibited LPS-induced autophagy in RAW264.7 macrophages. (A) Determination of BRF1 and autophagy related protein LC3,SQSTM1/p62, ATG7 and ATG5-ATG12 protein expression level after infection with Ad-GFP or Ad-BRF1 and LPS (500 ng/ml) in RAW264.7 macrophage for 24 h by Western blotting analysis. β-actin was regarded as controls corresponding to protein above. (B–E) Quantitative analysis of the bar graphs represented endogenous LC3(B), SQSTM1/p62 (C), ATG5-ATG12 (D) and ATG7 (E) expression compared to β-actin respectively. * P

    Journal: Genes & Diseases

    Article Title: BRF1 ameliorates LPS-induced inflammation through autophagy crosstalking with MAPK/ERK signaling

    doi: 10.1016/j.gendis.2018.04.004

    Figure Lengend Snippet: BRF1 inhibited LPS-induced autophagy in RAW264.7 macrophages. (A) Determination of BRF1 and autophagy related protein LC3,SQSTM1/p62, ATG7 and ATG5-ATG12 protein expression level after infection with Ad-GFP or Ad-BRF1 and LPS (500 ng/ml) in RAW264.7 macrophage for 24 h by Western blotting analysis. β-actin was regarded as controls corresponding to protein above. (B–E) Quantitative analysis of the bar graphs represented endogenous LC3(B), SQSTM1/p62 (C), ATG5-ATG12 (D) and ATG7 (E) expression compared to β-actin respectively. * P

    Article Snippet: 5% nonfat dry milk was used to reduce it nonspecific binding and primary antibodies was incubated overnight at 4 °C: Anti-SQSTM1 (p62), anti-caspase-12, anti-caspase-3, anti-ATG12, anti-BRF1, anti-ATG7, anti-β-actin, anti-ERK1/2, anti-p-ERK1/2 were bought from CST corresponding secondary antibody was incubated for 2 h at 25 °C .

    Techniques: Expressing, Infection, Western Blot

    The effect of BA on autophagic flux in KM3 cells. KM3 cells were treated with BA and LC3-II and P62 were applied to analyze the autophagic flux using Western blot analysis. The results showed that BA increased the amount of LC3-II and P62 in a dose-dependent

    Journal: Acta Pharmacologica Sinica

    Article Title: Betulinic acid inhibits autophagic flux and induces apoptosis in human multiple myeloma cells in vitro

    doi: 10.1038/aps.2012.102

    Figure Lengend Snippet: The effect of BA on autophagic flux in KM3 cells. KM3 cells were treated with BA and LC3-II and P62 were applied to analyze the autophagic flux using Western blot analysis. The results showed that BA increased the amount of LC3-II and P62 in a dose-dependent

    Article Snippet: The anti-LC3 and anti-P62 antibodies were purchased from Abcam, USA, and Santa Cruz Biotechnology, Inc, USA, respectively.

    Techniques: Western Blot

    Colocalization of PIM clusters with the autophagic machinery. Immunofluorescence images of representative HeLa cells 8 h after cluster induction by rapalog2 addition. mCherry (red), EGFP (green), and endogenous staining (cyan) of ubiquitin ( a ), p62 ( b ), NBR1 ( c ), LC3 ( d , e ), and LAMTOR4 ( f ) shown in inverted contrast. For e , HeLa cells were treated with 200 nM Bafilomcyin A1 for 8 h. Scale bars, 10 µm. Zooms show individual clusters, scale bar 2 µm. Plots show the fraction of yellow (yellow circles) and red (red squares) clusters colocalized with the marker per cell. Mean ± s.e.m. from 2 independent experiments with n = 21, 24, 24, 24, 26, and 22 cells for a – f

    Journal: Nature Communications

    Article Title: Probing aggrephagy using chemically-induced protein aggregates

    doi: 10.1038/s41467-018-06674-4

    Figure Lengend Snippet: Colocalization of PIM clusters with the autophagic machinery. Immunofluorescence images of representative HeLa cells 8 h after cluster induction by rapalog2 addition. mCherry (red), EGFP (green), and endogenous staining (cyan) of ubiquitin ( a ), p62 ( b ), NBR1 ( c ), LC3 ( d , e ), and LAMTOR4 ( f ) shown in inverted contrast. For e , HeLa cells were treated with 200 nM Bafilomcyin A1 for 8 h. Scale bars, 10 µm. Zooms show individual clusters, scale bar 2 µm. Plots show the fraction of yellow (yellow circles) and red (red squares) clusters colocalized with the marker per cell. Mean ± s.e.m. from 2 independent experiments with n = 21, 24, 24, 24, 26, and 22 cells for a – f

    Article Snippet: Primary antibodies used were mouse anti-p62 (Abcam, #ab56416, 1/2000), mouse anti-ubiquitin (Enzo, #BML-PW8810, 1/2000), and rabbit anti-GAPDH (Sigma, #G9545, 1/5000).

    Techniques: Immunofluorescence, Staining, Marker

    DEX promotes apoptosis by activating autophagy in MC3T3-E1 cells. (A–C) MC3T3-E1 cells were treated with DEX (1 μ M), 3-MA (5 mM) or 3-MA (5 mM) + DEX (1 μ M) and (A) the viability and (B and C) the apoptotic rate were measured using a Cell Counting Kit-8 assay. (D) Western blot analysis of apoptosis-associated proteins (PARP and caspase-3) and autophagy-associated proteins (P62, LC3B and Beclin1). (E and F) The expression of each protein normalized to the GAPDH was evaluated. Values are expressed as the mean ± standard deviation (n=3). * P

    Journal: International Journal of Molecular Medicine

    Article Title: Dexamethasone-induced production of reactive oxygen species promotes apoptosis via endoplasmic reticulum stress and autophagy in MC3T3-E1 cells

    doi: 10.3892/ijmm.2018.3412

    Figure Lengend Snippet: DEX promotes apoptosis by activating autophagy in MC3T3-E1 cells. (A–C) MC3T3-E1 cells were treated with DEX (1 μ M), 3-MA (5 mM) or 3-MA (5 mM) + DEX (1 μ M) and (A) the viability and (B and C) the apoptotic rate were measured using a Cell Counting Kit-8 assay. (D) Western blot analysis of apoptosis-associated proteins (PARP and caspase-3) and autophagy-associated proteins (P62, LC3B and Beclin1). (E and F) The expression of each protein normalized to the GAPDH was evaluated. Values are expressed as the mean ± standard deviation (n=3). * P

    Article Snippet: In addition, rabbit anti-Beclin1 (1:1,000 dilution, cat. no. ab62557), rabbit anti-LC3B (1:1,000 dilution, cat. no. ab51520), rabbit anti-P62 (1:1,000 dilution; cat. no. ab91526) and rabbit anti-cyclin-dependent kinase 2 (CDK2; 1:1,000 dilution; cat. no. ab32147) were obtained from Abcam (Cambridge, UK).

    Techniques: Cell Counting, Western Blot, Expressing, Standard Deviation

    ROS production induced by DEX promotes apoptosis in MC3T3-E1 cells through ER stress and autophagy. (A and B) ROS production rate of MC3T3-E1 cells and quantification of it, in the control, DEX (1 μ M), NAC (10 mM) and NAC (10 mM) + DEX (1 μ M) groups, as detected by flow cytometry. (C) Viability and (D and E) apoptosis after treatment with NAC. It was indicated that the activation of ROS was involved in the inhibition of viability induced by DEX. (F) Western blot analysis of autophagy-associated (LC3B, P62 and Beclin1) and ER stress-associated proteins (ATF4 and CHOP). (G and H) A relative quantification assay was performed to assess the expression of each protein normalized to the GAPDH expression Values are expressed as the mean ± standard deviation (n=3). * P

    Journal: International Journal of Molecular Medicine

    Article Title: Dexamethasone-induced production of reactive oxygen species promotes apoptosis via endoplasmic reticulum stress and autophagy in MC3T3-E1 cells

    doi: 10.3892/ijmm.2018.3412

    Figure Lengend Snippet: ROS production induced by DEX promotes apoptosis in MC3T3-E1 cells through ER stress and autophagy. (A and B) ROS production rate of MC3T3-E1 cells and quantification of it, in the control, DEX (1 μ M), NAC (10 mM) and NAC (10 mM) + DEX (1 μ M) groups, as detected by flow cytometry. (C) Viability and (D and E) apoptosis after treatment with NAC. It was indicated that the activation of ROS was involved in the inhibition of viability induced by DEX. (F) Western blot analysis of autophagy-associated (LC3B, P62 and Beclin1) and ER stress-associated proteins (ATF4 and CHOP). (G and H) A relative quantification assay was performed to assess the expression of each protein normalized to the GAPDH expression Values are expressed as the mean ± standard deviation (n=3). * P

    Article Snippet: In addition, rabbit anti-Beclin1 (1:1,000 dilution, cat. no. ab62557), rabbit anti-LC3B (1:1,000 dilution, cat. no. ab51520), rabbit anti-P62 (1:1,000 dilution; cat. no. ab91526) and rabbit anti-cyclin-dependent kinase 2 (CDK2; 1:1,000 dilution; cat. no. ab32147) were obtained from Abcam (Cambridge, UK).

    Techniques: Flow Cytometry, Cytometry, Activation Assay, Inhibition, Western Blot, Expressing, Standard Deviation

    Fluorescence intensity of autophagic vacuoles in MC3T3-E1 cells under fluorescence microscopy. (A) The intensity of autophagic vacuoles was evaluated by MDC staining (magnification, ×400; scale bar, 20 μ m) and (B) their average fluorescence intensity was quantified. It was indicated that DEX increased the number of autophagic vacuoles. (C) Western blot analysis was performed to determine the protein expression levels of autophagy-associated proteins Beclin1, LC3B and P62. (D) The relative expression of LC3B-II, Beclin1 and P62 were calculated. Values are expressed as the mean ± standard deviation (n=3). * P

    Journal: International Journal of Molecular Medicine

    Article Title: Dexamethasone-induced production of reactive oxygen species promotes apoptosis via endoplasmic reticulum stress and autophagy in MC3T3-E1 cells

    doi: 10.3892/ijmm.2018.3412

    Figure Lengend Snippet: Fluorescence intensity of autophagic vacuoles in MC3T3-E1 cells under fluorescence microscopy. (A) The intensity of autophagic vacuoles was evaluated by MDC staining (magnification, ×400; scale bar, 20 μ m) and (B) their average fluorescence intensity was quantified. It was indicated that DEX increased the number of autophagic vacuoles. (C) Western blot analysis was performed to determine the protein expression levels of autophagy-associated proteins Beclin1, LC3B and P62. (D) The relative expression of LC3B-II, Beclin1 and P62 were calculated. Values are expressed as the mean ± standard deviation (n=3). * P

    Article Snippet: In addition, rabbit anti-Beclin1 (1:1,000 dilution, cat. no. ab62557), rabbit anti-LC3B (1:1,000 dilution, cat. no. ab51520), rabbit anti-P62 (1:1,000 dilution; cat. no. ab91526) and rabbit anti-cyclin-dependent kinase 2 (CDK2; 1:1,000 dilution; cat. no. ab32147) were obtained from Abcam (Cambridge, UK).

    Techniques: Fluorescence, Microscopy, Staining, Western Blot, Expressing, Standard Deviation

    Effect of SVIP and starvation on cell viability and autophagy in CCl 4 -treated HepG2 cells. a MTT assay showing the viability of HepG2 cells transfected with SVIP-expressing plasmid or vector control treated with CCl 4 or DMSO for 0–72 h. b Western blots (left panel) showing the expression of SVIP, Beclin1, p62, and LC3B in HepG2 cells transfected with siSVIP or siRNA control treated with CCl 4 or DMSO for 24 h. MTT assays (right panel) showing the viability of HepG2 cells transfected with siSVIP or siRNA control treated with CCl 4 or DMSO for 0–72 h. c Western blots showing the expression of SVIP, Beclin1, p62, and LC3B in HepG2 cells treated with CCl 4 or DMSO (12 h) in the absence and presence of starvation (2 h). d Western blots (left panel) and MTT assays (right panel) showing SVIP, p62, Beclin1, and LC3B expression and cell viability of CCl 4 -treated HepG2 cells transfected with SVIP expressing plasmid or vector in the presence or absence of starvation. Starvation started at 12, 36, and 60 h as indicated by arrows. e Western blots (left panel) and MTT assays (right panel) showing the expression of SVIP, Beclin1, p62, and LC3B in CCl 4 -treated HepG2 cells transfected with siSVIP or siRNA control in the absence and presence of starvation. 72 h after siRNA transfection, cells were subjected to CCl 4 treatment in the absence and presence of starvation (45 min). Starvation started at 12 and 36 h as indicated by arrows. Data are presented as mean ± SD in three independent experiments. LC3-II/LC3-I ratio represents the autophagy flux. * p

    Journal: Cell Death & Disease

    Article Title: SVIP alleviates CCl4-induced liver fibrosis via activating autophagy and protecting hepatocytes

    doi: 10.1038/s41419-019-1311-0

    Figure Lengend Snippet: Effect of SVIP and starvation on cell viability and autophagy in CCl 4 -treated HepG2 cells. a MTT assay showing the viability of HepG2 cells transfected with SVIP-expressing plasmid or vector control treated with CCl 4 or DMSO for 0–72 h. b Western blots (left panel) showing the expression of SVIP, Beclin1, p62, and LC3B in HepG2 cells transfected with siSVIP or siRNA control treated with CCl 4 or DMSO for 24 h. MTT assays (right panel) showing the viability of HepG2 cells transfected with siSVIP or siRNA control treated with CCl 4 or DMSO for 0–72 h. c Western blots showing the expression of SVIP, Beclin1, p62, and LC3B in HepG2 cells treated with CCl 4 or DMSO (12 h) in the absence and presence of starvation (2 h). d Western blots (left panel) and MTT assays (right panel) showing SVIP, p62, Beclin1, and LC3B expression and cell viability of CCl 4 -treated HepG2 cells transfected with SVIP expressing plasmid or vector in the presence or absence of starvation. Starvation started at 12, 36, and 60 h as indicated by arrows. e Western blots (left panel) and MTT assays (right panel) showing the expression of SVIP, Beclin1, p62, and LC3B in CCl 4 -treated HepG2 cells transfected with siSVIP or siRNA control in the absence and presence of starvation. 72 h after siRNA transfection, cells were subjected to CCl 4 treatment in the absence and presence of starvation (45 min). Starvation started at 12 and 36 h as indicated by arrows. Data are presented as mean ± SD in three independent experiments. LC3-II/LC3-I ratio represents the autophagy flux. * p

    Article Snippet: Primary antibodies, polyclonal rabbit anti-LC3B was purchased from CST (USA), anti-p62, and Beclin1 were purchased from Santa Cruz Biotechnology, USA.

    Techniques: MTT Assay, Transfection, Expressing, Plasmid Preparation, Western Blot

    Starvation increasing SVIP expression alleviated CCl 4 -induced liver fibrosis. Twenty SD rats were injected subcutaneously with 1 ml/kg 50% (v/v) CCl 4 or olive oil (control, n = 3, 0 death) twice a week for 8 weeks, CCl 4 -treated rats were starved for 24 h twice weekly (stv24hx2/w CCl4, n = 6, 1 death) or 48 h once weekly (stv48hx1/w CCl 4 , n = 5, 0 death) or without starvation (CCl 4 , n = 6, 2 death), the liver tissues were subjected to the following assays. a Masson’s trichrome staining of liver sections from rats exposed to CCl 4 or olive oil (left panel) and the proportions of collagenic areas on Masson’s trichrome-stained sections (right panel). b Western blots showing protein expression of SVIP, Beclin1, p62, and LC3 in liver tissues (left panel) and quantification of relative protein levels (right panel) calibrated with β-actin. c Immunohistochemistry (IHC) analysis of SVIP, p62, and Beclin1 expression in liver sections. d PCR analysis of mRNA levels of SVIP, p62, LC3B, and Atg5. Relative mRNA levels were calibrated with β-actin. Data are presented as mean ± SD, * p

    Journal: Cell Death & Disease

    Article Title: SVIP alleviates CCl4-induced liver fibrosis via activating autophagy and protecting hepatocytes

    doi: 10.1038/s41419-019-1311-0

    Figure Lengend Snippet: Starvation increasing SVIP expression alleviated CCl 4 -induced liver fibrosis. Twenty SD rats were injected subcutaneously with 1 ml/kg 50% (v/v) CCl 4 or olive oil (control, n = 3, 0 death) twice a week for 8 weeks, CCl 4 -treated rats were starved for 24 h twice weekly (stv24hx2/w CCl4, n = 6, 1 death) or 48 h once weekly (stv48hx1/w CCl 4 , n = 5, 0 death) or without starvation (CCl 4 , n = 6, 2 death), the liver tissues were subjected to the following assays. a Masson’s trichrome staining of liver sections from rats exposed to CCl 4 or olive oil (left panel) and the proportions of collagenic areas on Masson’s trichrome-stained sections (right panel). b Western blots showing protein expression of SVIP, Beclin1, p62, and LC3 in liver tissues (left panel) and quantification of relative protein levels (right panel) calibrated with β-actin. c Immunohistochemistry (IHC) analysis of SVIP, p62, and Beclin1 expression in liver sections. d PCR analysis of mRNA levels of SVIP, p62, LC3B, and Atg5. Relative mRNA levels were calibrated with β-actin. Data are presented as mean ± SD, * p

    Article Snippet: Primary antibodies, polyclonal rabbit anti-LC3B was purchased from CST (USA), anti-p62, and Beclin1 were purchased from Santa Cruz Biotechnology, USA.

    Techniques: Expressing, Injection, Staining, Western Blot, Immunohistochemistry, Polymerase Chain Reaction

    SVIP regulates autophagy in HepG2 cells. a Western blots (WB) and b PCR showing the expression of Beclin1, p62, LC3B, Atg5 in HepG2 cells transfected with SVIP expressing or control plasmid. c and d Western blots showing the expression of TFEB, Rab7, and conversion of LC3BII to LC3BI ( c ) in cells transfected with SVIP expressing or/and control plasmid in different dose ( d ). HepG2 cells were transfected with 2 μg total DNA, containing SVIP-expressing plasmid or vector control or 1 μg SVIP-expressing plasmid + 1 μg vector control. Baf-A1, (100 ng/ml) Bafilomycin A1. e Western blots and f PCR showing the expression of Beclin1, p62, LC3B, Atg5 in silence SVIP HepG2 cells. 24 and 72 h after the plasmid and siRNA transfection, cells were subjected to WB and reverse-transcriptional PCR, respectively. Bar graph indicates the bands’ intensity of mRNA calibrated with β-actin RNA level. LC3-II/LC3-I ratio represents the autophagy flux. Data are presented as mean ± standard division (SD) in three independent experiments. * p

    Journal: Cell Death & Disease

    Article Title: SVIP alleviates CCl4-induced liver fibrosis via activating autophagy and protecting hepatocytes

    doi: 10.1038/s41419-019-1311-0

    Figure Lengend Snippet: SVIP regulates autophagy in HepG2 cells. a Western blots (WB) and b PCR showing the expression of Beclin1, p62, LC3B, Atg5 in HepG2 cells transfected with SVIP expressing or control plasmid. c and d Western blots showing the expression of TFEB, Rab7, and conversion of LC3BII to LC3BI ( c ) in cells transfected with SVIP expressing or/and control plasmid in different dose ( d ). HepG2 cells were transfected with 2 μg total DNA, containing SVIP-expressing plasmid or vector control or 1 μg SVIP-expressing plasmid + 1 μg vector control. Baf-A1, (100 ng/ml) Bafilomycin A1. e Western blots and f PCR showing the expression of Beclin1, p62, LC3B, Atg5 in silence SVIP HepG2 cells. 24 and 72 h after the plasmid and siRNA transfection, cells were subjected to WB and reverse-transcriptional PCR, respectively. Bar graph indicates the bands’ intensity of mRNA calibrated with β-actin RNA level. LC3-II/LC3-I ratio represents the autophagy flux. Data are presented as mean ± standard division (SD) in three independent experiments. * p

    Article Snippet: Primary antibodies, polyclonal rabbit anti-LC3B was purchased from CST (USA), anti-p62, and Beclin1 were purchased from Santa Cruz Biotechnology, USA.

    Techniques: Western Blot, Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation

    Starvation enhanced autophagy but had no effect on SVIP expression in HepG2 cells and in mice. a Western blots (WB) and b PCR showing the expression of SIVP as well as Beclin1, p62, LC3B, Atg5. HepG2 cells were maintained in HBSS for 2 h for starvation. Data are presented as mean ± SD in three independent experiments. LC3-II/LC3-I ratio represents the autophagy flux. c and d Western blots (WB) and PCR showing the expression of SIVP, as well as Beclin1, p62, and LC3B in mouse liver ( c ) or brain ( d ). Mice were starved for an indicated period ( n = 3). Bar graph indicates the band’ intensity calibrated with β-actin. * p

    Journal: Cell Death & Disease

    Article Title: SVIP alleviates CCl4-induced liver fibrosis via activating autophagy and protecting hepatocytes

    doi: 10.1038/s41419-019-1311-0

    Figure Lengend Snippet: Starvation enhanced autophagy but had no effect on SVIP expression in HepG2 cells and in mice. a Western blots (WB) and b PCR showing the expression of SIVP as well as Beclin1, p62, LC3B, Atg5. HepG2 cells were maintained in HBSS for 2 h for starvation. Data are presented as mean ± SD in three independent experiments. LC3-II/LC3-I ratio represents the autophagy flux. c and d Western blots (WB) and PCR showing the expression of SIVP, as well as Beclin1, p62, and LC3B in mouse liver ( c ) or brain ( d ). Mice were starved for an indicated period ( n = 3). Bar graph indicates the band’ intensity calibrated with β-actin. * p

    Article Snippet: Primary antibodies, polyclonal rabbit anti-LC3B was purchased from CST (USA), anti-p62, and Beclin1 were purchased from Santa Cruz Biotechnology, USA.

    Techniques: Expressing, Mouse Assay, Western Blot, Polymerase Chain Reaction

    Dynamics of autophagy and SVIP expression in HepG2 cells treated with CCl 4 or DMSO for an indicated period. a Protein expression of p62, LC3B, and SVIP changed according to the period of CCl 4 treatment in HepG2 cells was detected by WB. b The mRNA ratios of CCl 4 -treated to vehicle-treated cells of each gene in HepG2 cells, analyzed using reverse transcription-PCR. Data are presented as mean ± SD in three independent experiments. * p

    Journal: Cell Death & Disease

    Article Title: SVIP alleviates CCl4-induced liver fibrosis via activating autophagy and protecting hepatocytes

    doi: 10.1038/s41419-019-1311-0

    Figure Lengend Snippet: Dynamics of autophagy and SVIP expression in HepG2 cells treated with CCl 4 or DMSO for an indicated period. a Protein expression of p62, LC3B, and SVIP changed according to the period of CCl 4 treatment in HepG2 cells was detected by WB. b The mRNA ratios of CCl 4 -treated to vehicle-treated cells of each gene in HepG2 cells, analyzed using reverse transcription-PCR. Data are presented as mean ± SD in three independent experiments. * p

    Article Snippet: Primary antibodies, polyclonal rabbit anti-LC3B was purchased from CST (USA), anti-p62, and Beclin1 were purchased from Santa Cruz Biotechnology, USA.

    Techniques: Expressing, Western Blot, Polymerase Chain Reaction

    Effect of CCl 4 treatment on SVIP expression, autophagy and histological alterations. The samples of olive oil ( n = 3) or CCl 4 injected Sprague–Dawley (SD) rats for 3, 5, and 8 weeks ( n = 6) were subjected to the following assays. a Masson’s trichrome staining for evaluation of collagen deposition. b Representative hematoxylin–eosin (H E)-stained liver sections from rats exposed to CCl 4 for 3, 5, or 8 weeks for evaluation of steatosis. c The proportions of collagenic (left panel) and steatotic (right panel) areas on Masson’s trichrome-stained and H E-stained liver sections, respectively. d Left panel: representative enzymatic activity of ALT and AST in blood plasma of rats exposed to CCl 4 for 3, 5, or 8 weeks, right panel: the ratio between AST and ALT. e Immunohistochemistry (IHC) analysis of SVIP as well as α-SMA, p62, and Beclin1 expression in liver sections. f and g Quantitative expressions of SVIP, LC3, p62/SQSTM1, Atg5, and Beclin1 in rat livers were analyzed by WB ( f ) and PCR ( g ). Bar graph indicates the bands’ intensity calibrated with β-actin. Data were presented as mean ± SD, n = 3 or 6. * p

    Journal: Cell Death & Disease

    Article Title: SVIP alleviates CCl4-induced liver fibrosis via activating autophagy and protecting hepatocytes

    doi: 10.1038/s41419-019-1311-0

    Figure Lengend Snippet: Effect of CCl 4 treatment on SVIP expression, autophagy and histological alterations. The samples of olive oil ( n = 3) or CCl 4 injected Sprague–Dawley (SD) rats for 3, 5, and 8 weeks ( n = 6) were subjected to the following assays. a Masson’s trichrome staining for evaluation of collagen deposition. b Representative hematoxylin–eosin (H E)-stained liver sections from rats exposed to CCl 4 for 3, 5, or 8 weeks for evaluation of steatosis. c The proportions of collagenic (left panel) and steatotic (right panel) areas on Masson’s trichrome-stained and H E-stained liver sections, respectively. d Left panel: representative enzymatic activity of ALT and AST in blood plasma of rats exposed to CCl 4 for 3, 5, or 8 weeks, right panel: the ratio between AST and ALT. e Immunohistochemistry (IHC) analysis of SVIP as well as α-SMA, p62, and Beclin1 expression in liver sections. f and g Quantitative expressions of SVIP, LC3, p62/SQSTM1, Atg5, and Beclin1 in rat livers were analyzed by WB ( f ) and PCR ( g ). Bar graph indicates the bands’ intensity calibrated with β-actin. Data were presented as mean ± SD, n = 3 or 6. * p

    Article Snippet: Primary antibodies, polyclonal rabbit anti-LC3B was purchased from CST (USA), anti-p62, and Beclin1 were purchased from Santa Cruz Biotechnology, USA.

    Techniques: Expressing, Injection, Staining, Activity Assay, AST Assay, Immunohistochemistry, Western Blot, Polymerase Chain Reaction

    Interfering with p62 protein level or function increases cell death induced by mutant huntingtin. (A) GFP-positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h of expression, GFP-positive cells attached to the surface with a normal nuclear appearance (open arrowheads) and were scored as live, whereas rounded, partially detached cells with a condensed or fragmented nuclei (arrows) were scored as dead. More than 200 GFP-positive cells in randomly selected microscope views were scored. Bars, 20 μm. (B) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N-HttQ68–induced cell death in HeLa and SHSY-5Y neuroblastoma cells.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: Interfering with p62 protein level or function increases cell death induced by mutant huntingtin. (A) GFP-positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h of expression, GFP-positive cells attached to the surface with a normal nuclear appearance (open arrowheads) and were scored as live, whereas rounded, partially detached cells with a condensed or fragmented nuclei (arrows) were scored as dead. More than 200 GFP-positive cells in randomly selected microscope views were scored. Bars, 20 μm. (B) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N-HttQ68–induced cell death in HeLa and SHSY-5Y neuroblastoma cells.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Mutagenesis, Staining, Expressing, Microscopy, Construct

    Both the PB1 and UBA domains are needed for p62 to form cytoplasmic bodies. (A) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts. Asterisks indicate point mutations in the PB1 (D69A) and UBA (I431A) domains, respectively. (B) S–GFP-p62 cells were transiently transfected with the indicated myc-tagged p62 constructs, fixed, and stained for the myc tag. Bars, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: Both the PB1 and UBA domains are needed for p62 to form cytoplasmic bodies. (A) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts. Asterisks indicate point mutations in the PB1 (D69A) and UBA (I431A) domains, respectively. (B) S–GFP-p62 cells were transiently transfected with the indicated myc-tagged p62 constructs, fixed, and stained for the myc tag. Bars, 20 μm.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Construct, Transfection, Staining

    p62 forms a shell around huntingtin protein aggregates. (A) HeLa cells transiently expressing a Flag-tagged NH 2 -terminal fragment (amino acids 1–171) of huntingtin containing a 68-polyglutamine expansion, Flag–N-HttQ68. After 48 h, the cells were fixed, and Flag–N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was stained red using the p62 pAb. (B) Endogenous p62 forms a shell around aggregated GFP–N-HttQ68. Transiently transfected HeLa cells were fixed 48 h after transfection, and endogenous p62 was detected using a p62 mAb (red). (C and D) The p62 shell surrounding GFP–N-HttQ68 was detected independently of the use of antibodies. (C) GFP–N-HttQ68 and DsRed-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 48 h of expression. The left panel shows one confocal plane of a representative GFP–N-HttQ68 and DsRed-p62 double-positive structure, and the right panel shows the Z-stack of planes with side views of the aggregate at the side and at the top. N, nucleus. (A–C) Nuclei were detected using Draq5. (D) GFP–N-HttQ68 and tdTomato-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 24 h of expression. (E) Transiently expressed Flag–N-HttQ68 and myc-LC3 colocalize in GFP-p62–positive structures. S–GFP-p62 cells were fixed 48 h after transfection, Flag–N-HttQ68 was stained red using a Flag tag mAb, and myc-LC3 was stained far red (blue) using a chicken myc tag pAb (top). The bottom panel shows a live cell image of a GFP–N-HttQ68 aggregate surrounded by a shell containing tdTomato-LC3. Bars, 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: p62 forms a shell around huntingtin protein aggregates. (A) HeLa cells transiently expressing a Flag-tagged NH 2 -terminal fragment (amino acids 1–171) of huntingtin containing a 68-polyglutamine expansion, Flag–N-HttQ68. After 48 h, the cells were fixed, and Flag–N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was stained red using the p62 pAb. (B) Endogenous p62 forms a shell around aggregated GFP–N-HttQ68. Transiently transfected HeLa cells were fixed 48 h after transfection, and endogenous p62 was detected using a p62 mAb (red). (C and D) The p62 shell surrounding GFP–N-HttQ68 was detected independently of the use of antibodies. (C) GFP–N-HttQ68 and DsRed-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 48 h of expression. The left panel shows one confocal plane of a representative GFP–N-HttQ68 and DsRed-p62 double-positive structure, and the right panel shows the Z-stack of planes with side views of the aggregate at the side and at the top. N, nucleus. (A–C) Nuclei were detected using Draq5. (D) GFP–N-HttQ68 and tdTomato-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 24 h of expression. (E) Transiently expressed Flag–N-HttQ68 and myc-LC3 colocalize in GFP-p62–positive structures. S–GFP-p62 cells were fixed 48 h after transfection, Flag–N-HttQ68 was stained red using a Flag tag mAb, and myc-LC3 was stained far red (blue) using a chicken myc tag pAb (top). The bottom panel shows a live cell image of a GFP–N-HttQ68 aggregate surrounded by a shell containing tdTomato-LC3. Bars, 5 μm.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Expressing, Staining, Transfection, FLAG-tag

    p62 bodies are degraded by autophagy. (A) Comparison of p62 degradation using pulse-chase labeling with 35 S-methionine and immunoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed with 35 S-methionine and incubated for the indicated times in nonradioactive medium. After immunopurification, the amount of radioactive p62 was determined by autoradiography, and the total amount of p62 was determined by an immunoblot of the same membrane. The p62 level in total cellular lysates after different times of 10 μg/ml cycloheximide treatment determined by immunoblotting (bottom). (B) The levels of p62 and LC3-II change with autophagic activity. HeLa or S–GFP-p62 cells were either left untreated or rapamycin (10 μg/ml) or bafilomycin A1 (Baf. A1; 10 μg/ml) was added for 18 h. Immunoblots were sequentially probed using LC3, p62, and actin antibodies. (C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S–GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control. (E) The majority of S–GFP-p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3. S–GFP-p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained red using an anti-myc tag mAb. The boxed area indicates the part of the cell that is shown to the left at a higher magnification. Bars, 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: p62 bodies are degraded by autophagy. (A) Comparison of p62 degradation using pulse-chase labeling with 35 S-methionine and immunoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed with 35 S-methionine and incubated for the indicated times in nonradioactive medium. After immunopurification, the amount of radioactive p62 was determined by autoradiography, and the total amount of p62 was determined by an immunoblot of the same membrane. The p62 level in total cellular lysates after different times of 10 μg/ml cycloheximide treatment determined by immunoblotting (bottom). (B) The levels of p62 and LC3-II change with autophagic activity. HeLa or S–GFP-p62 cells were either left untreated or rapamycin (10 μg/ml) or bafilomycin A1 (Baf. A1; 10 μg/ml) was added for 18 h. Immunoblots were sequentially probed using LC3, p62, and actin antibodies. (C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S–GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control. (E) The majority of S–GFP-p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3. S–GFP-p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained red using an anti-myc tag mAb. The boxed area indicates the part of the cell that is shown to the left at a higher magnification. Bars, 5 μm.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Pulse Chase, Labeling, Incubation, Immu-Puri, Autoradiography, Activity Assay, Western Blot, Inhibition, Staining, Imaging, Transfection

    p62 bodies are found both as membrane-free protein aggregates and as membrane-confined autophagosomal and lysosomal structures. (A) Localization of bodies containing endogenous p62 or transiently expressed GFP-p62 relative to EEA1-positive early endosomes. Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488, and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555. Alternatively, transiently expressed GFP-p62 was expressed in HeLa cells, and EEA1 was stained red with EEA1 mAb (bottom). The boxed area is shown to the right at a higher magnification. (B) Colocalization of GFP-p62 and CD63 (stained red using a mAb) in HeLa cells stably expressing GFP-p62. (C) Immunoelectron micrograph of S–GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads). (D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: p62 bodies are found both as membrane-free protein aggregates and as membrane-confined autophagosomal and lysosomal structures. (A) Localization of bodies containing endogenous p62 or transiently expressed GFP-p62 relative to EEA1-positive early endosomes. Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488, and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555. Alternatively, transiently expressed GFP-p62 was expressed in HeLa cells, and EEA1 was stained red with EEA1 mAb (bottom). The boxed area is shown to the right at a higher magnification. (B) Colocalization of GFP-p62 and CD63 (stained red using a mAb) in HeLa cells stably expressing GFP-p62. (C) Immunoelectron micrograph of S–GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads). (D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Staining, Labeling, Stable Transfection, Expressing

    p62 is found both in autophagosomes and in cytosolic aggregates/sequestosomes. (A) Immuno-EM of GFP-p62. S–GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A–gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo). (B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1. (C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: p62 is found both in autophagosomes and in cytosolic aggregates/sequestosomes. (A) Immuno-EM of GFP-p62. S–GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A–gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo). (B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1. (C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Labeling, Immunofluorescence, Transfection

    The p62 bodies in the cytoplasm of HeLa cells are ubiquitin-containing protein aggregates. (A) HeLa cells were fixed and stained for p62. The different patterns of p62 distribution were scored in > 200 cells as cytoplasmic with small bodies, cytoplasmic with large and intense bodies, equal nuclear and cytoplasmic staining, and nuclear enriched. The percentage of cells in the respective groups is indicated. (B) Expression level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S–GFP-p62) compared with the level of endogenous p62 in the parent HeLa cells. (C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate localization of GFP-tagged p62 similar to endogenous p62. Video microscopy of live cells demonstrated that the small, faint bodies (thin arrows) displayed directed migration, whereas the majority of the larger, intense bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1 ). A still image from Video 1 is shown here. (D) Bodies containing either endogenous p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin. HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1) mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488 (green), respectively. Cells expressing GFP-p62 were only stained for polyubiquitin (red). Bars, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: The p62 bodies in the cytoplasm of HeLa cells are ubiquitin-containing protein aggregates. (A) HeLa cells were fixed and stained for p62. The different patterns of p62 distribution were scored in > 200 cells as cytoplasmic with small bodies, cytoplasmic with large and intense bodies, equal nuclear and cytoplasmic staining, and nuclear enriched. The percentage of cells in the respective groups is indicated. (B) Expression level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S–GFP-p62) compared with the level of endogenous p62 in the parent HeLa cells. (C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate localization of GFP-tagged p62 similar to endogenous p62. Video microscopy of live cells demonstrated that the small, faint bodies (thin arrows) displayed directed migration, whereas the majority of the larger, intense bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1 ). A still image from Video 1 is shown here. (D) Bodies containing either endogenous p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin. HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1) mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488 (green), respectively. Cells expressing GFP-p62 were only stained for polyubiquitin (red). Bars, 20 μm.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Staining, Expressing, Stable Transfection, Microscopy, Migration, Transfection

    p62 is required for the formation of GFP-LC3–positive punctuated structures in HeLa cells. (A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3–positive cells with punctuate localization are indicated to the right. (B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.

    Journal: The Journal of Cell Biology

    Article Title: p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

    doi: 10.1083/jcb.200507002

    Figure Lengend Snippet: p62 is required for the formation of GFP-LC3–positive punctuated structures in HeLa cells. (A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3–positive cells with punctuate localization are indicated to the right. (B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-p62 and EEA1 mAbs (BD Transduction Laboratories); p62 pAb (Progen Biotechnik); polyubiquitin mAb (clone FK1; Affinity BioReagents, Inc.); CD63 mAbs (Developmental Studies Hybridoma Bank, University of Iowa); anti-GFP antibody (Ab290; Abcam Ltd.); anti-EGF receptor and myc-tag antibodies (9E10; Santa Cruz Biotechnology, Inc.); antiactin antibody (Sigma-Aldrich); and LC3 antibody (gift from T. Yoshimori, National Institute of Genetics, Shizuoka, Japan; ).

    Techniques: Transfection, Staining, Mutagenesis, Immunoprecipitation, Construct

    RNF216 regulated GN11 cells migration through autophagy. (A) Depletion of RNF216 upregulated autophagy flux in GN11 cells. The protein levels of LC3 and P62 were detected with immunoblotting in GN11 cells transfected with siNC and siRNF. ACTIN was used as a loading control. (B,C) Quantification of LC3-II (B) and P62 (C) protein levels in GN11 cells as detected by immunoblotting. Data is shown as the mean ± SEM of three independent experiments, * P

    Journal: Frontiers in Endocrinology

    Article Title: RNF216 Regulates the Migration of Immortalized GnRH Neurons by Suppressing Beclin1-Mediated Autophagy

    doi: 10.3389/fendo.2019.00012

    Figure Lengend Snippet: RNF216 regulated GN11 cells migration through autophagy. (A) Depletion of RNF216 upregulated autophagy flux in GN11 cells. The protein levels of LC3 and P62 were detected with immunoblotting in GN11 cells transfected with siNC and siRNF. ACTIN was used as a loading control. (B,C) Quantification of LC3-II (B) and P62 (C) protein levels in GN11 cells as detected by immunoblotting. Data is shown as the mean ± SEM of three independent experiments, * P

    Article Snippet: The membranes were blocked with 5% fat-free milk in TBS with 0.1% Tween-20 for 1 h. The primary antibodies used were as following: 1:2,000 rabbit anti-RNF216 (Sigma, St. Louis, MO, USA); 1:2,000 rabbit anti-BECN1 (Cell Signaling Technology, Danvers, MA, USA); 1:4,000 rabbit anti-P62 (Cell Signaling Technology, Danvers, MA, USA); 1:1,000 rabbit anti-LC3 (Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, Transfection

    Drugs influence the autophagic flux in LTC cells. CHC and CHC plus metfomin (Met) treatment caused accumulation of p62 whereas metformin treatment reduced p62 level (A) . Gemcitabine (Gem) treatment reduced the p62 level (B) .

    Journal: Frontiers in Oncology

    Article Title: Blocking Autophagy in Cancer-Associated Fibroblasts Supports Chemotherapy of Pancreatic Cancer Cells

    doi: 10.3389/fonc.2018.00590

    Figure Lengend Snippet: Drugs influence the autophagic flux in LTC cells. CHC and CHC plus metfomin (Met) treatment caused accumulation of p62 whereas metformin treatment reduced p62 level (A) . Gemcitabine (Gem) treatment reduced the p62 level (B) .

    Article Snippet: Primary antibodies against type I collagen (collagen I, code ab34710), p62 (code ab109012-100), and β-actin (code A5441) were obtained from Abcam (Cambridge, UK) or Sigma-Aldrich.

    Techniques:

    p62 does not allow Tax autophagic degradation at the steady-state. ( a ) HeLa cells stably expressing GFP-LC3 were transfected with Tax-His and analyzed by confocal microscopy after staining with His- (white) and p62- or OPTN-specific (red) antibodies. Representative images are shown. Tax, p62 or OPTN and GFP-LC3 signals were quantified along the segment represented on the merge panel and plotted on the histogram. Scale bar = 10 μm. ( b , c ) HeLa cells transiently expressing Tax-His were treated with lysosomal inhibitors ( b , E64D/Pep.) or starved ( c , HBSS) before lysis, western blot analyses and quantification. Full-length blots are presented in Supplementary Fig. S4 . Blots and graphs show results representative of at least 3 experiments.

    Journal: Scientific Reports

    Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function

    doi: 10.1038/s41598-019-52408-x

    Figure Lengend Snippet: p62 does not allow Tax autophagic degradation at the steady-state. ( a ) HeLa cells stably expressing GFP-LC3 were transfected with Tax-His and analyzed by confocal microscopy after staining with His- (white) and p62- or OPTN-specific (red) antibodies. Representative images are shown. Tax, p62 or OPTN and GFP-LC3 signals were quantified along the segment represented on the merge panel and plotted on the histogram. Scale bar = 10 μm. ( b , c ) HeLa cells transiently expressing Tax-His were treated with lysosomal inhibitors ( b , E64D/Pep.) or starved ( c , HBSS) before lysis, western blot analyses and quantification. Full-length blots are presented in Supplementary Fig. S4 . Blots and graphs show results representative of at least 3 experiments.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).

    Techniques: Stable Transfection, Expressing, Transfection, Confocal Microscopy, Staining, Lysis, Western Blot

    p62 potentiates Tax-dependent NF-κB activation upstream of IKK activation. ( a ) Wild type (WT) and p62 −/− MEF cells were transfected with Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. ( b ) WT and p62 −/− MEF cells were transfected with Tax-His. After fractionation of cell nuclei, transcriptionally active p65 was quantified by ELISA. ( c ) Lysates from WT and p62 −/− MEF cells transfected with Tax-His were analyzed by western blot. ( d ) WT and p62 −/− MEF cells were transfected with Tax-His. After RNA extraction and conversion to cDNAs, Il6 and gapdh cDNAs were amplified by PCR (left panel). The normalized Il6 signal intensities were calculated and are shown relative to the corresponding Tax-negative conditions on the graph (results from 2 independent experiments). Cell lysates were also analyzed by western blot (right panel). ( e ) HEK293T cells were transfected with control (siCTRL) or p62 -specific (si p62 ) siRNA and Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. ( f ) Lysates from HEK293T cells transfected with siCTRL or si p62 and Tax-His were analyzed by western blot. ( g ) Jurkat cells were transfected with increasing amounts of Myc-p62 and an NF-κB-luc construct, followed by transduction with an empty or Flag-Tax-encoding lentivector. Luciferase activity was measured and normalized to the corresponding Tax-negative condition. Values obtained with endogenous p62 were set to 1 and other values are shown as fold change over the “endogenous p62” condition. The graph shows results from 3 independent experiments. ( h ) Lysates from Jurkat cells were analyzed by western blot. ***p

    Journal: Scientific Reports

    Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function

    doi: 10.1038/s41598-019-52408-x

    Figure Lengend Snippet: p62 potentiates Tax-dependent NF-κB activation upstream of IKK activation. ( a ) Wild type (WT) and p62 −/− MEF cells were transfected with Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. ( b ) WT and p62 −/− MEF cells were transfected with Tax-His. After fractionation of cell nuclei, transcriptionally active p65 was quantified by ELISA. ( c ) Lysates from WT and p62 −/− MEF cells transfected with Tax-His were analyzed by western blot. ( d ) WT and p62 −/− MEF cells were transfected with Tax-His. After RNA extraction and conversion to cDNAs, Il6 and gapdh cDNAs were amplified by PCR (left panel). The normalized Il6 signal intensities were calculated and are shown relative to the corresponding Tax-negative conditions on the graph (results from 2 independent experiments). Cell lysates were also analyzed by western blot (right panel). ( e ) HEK293T cells were transfected with control (siCTRL) or p62 -specific (si p62 ) siRNA and Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. ( f ) Lysates from HEK293T cells transfected with siCTRL or si p62 and Tax-His were analyzed by western blot. ( g ) Jurkat cells were transfected with increasing amounts of Myc-p62 and an NF-κB-luc construct, followed by transduction with an empty or Flag-Tax-encoding lentivector. Luciferase activity was measured and normalized to the corresponding Tax-negative condition. Values obtained with endogenous p62 were set to 1 and other values are shown as fold change over the “endogenous p62” condition. The graph shows results from 3 independent experiments. ( h ) Lysates from Jurkat cells were analyzed by western blot. ***p

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).

    Techniques: Activation Assay, Transfection, Construct, Luciferase, Activity Assay, Fractionation, Enzyme-linked Immunosorbent Assay, Western Blot, RNA Extraction, Amplification, Polymerase Chain Reaction, Transduction

    Involvement of ubiquitin binding by p62 in Tax-induced NF-κB activation: working model. p62 associates with Tax in peri-golgian structures, downstream of the formation of Tax/IKK complexes, and participates in the efficient activation of the IKK complex by a mechanism requiring binding to ubiquitin chains. See text for further details. T1BP1: TAX1BP1. Artwork is a derivative of Servier Medical Art ( https://smart.servier.com/ ), used under CC BY 3.0 FR ( https://creativecommons.org/licenses/by/3.0/ ), and includes modifications in shapes, colors and disposition of the original material.

    Journal: Scientific Reports

    Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function

    doi: 10.1038/s41598-019-52408-x

    Figure Lengend Snippet: Involvement of ubiquitin binding by p62 in Tax-induced NF-κB activation: working model. p62 associates with Tax in peri-golgian structures, downstream of the formation of Tax/IKK complexes, and participates in the efficient activation of the IKK complex by a mechanism requiring binding to ubiquitin chains. See text for further details. T1BP1: TAX1BP1. Artwork is a derivative of Servier Medical Art ( https://smart.servier.com/ ), used under CC BY 3.0 FR ( https://creativecommons.org/licenses/by/3.0/ ), and includes modifications in shapes, colors and disposition of the original material.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).

    Techniques: Binding Assay, Activation Assay

    p62 associates with Tax/IKK signalosomes in peri-golgian structures. ( a , b ) Jurkat cells transiently expressing Tax-His and HTLV-1 chronically infected C91PL cells were analyzed by confocal microscopy after staining with His- or Tax- (green), p62- (red), and GM130- ( a , white) and IKKγ-specific ( b , white) antibodies. Nuclei were counterstained with DAPI (blue). Representative images are shown. Tax, p62, GM130 and IKKγ signals were quantified along the segments represented on the merge panels and plotted on the histogram. Scale bar = 10 μm. ( c ) Lysates from HeLa cells transiently expressing Tax-His and/or IKKγ-FLAG were submitted to a FLAG-immunoprecipitation followed by a His-specific Ni-NTA purification and western blot analyses. Full-length blots are presented in Supplementary Fig. S4 .

    Journal: Scientific Reports

    Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function

    doi: 10.1038/s41598-019-52408-x

    Figure Lengend Snippet: p62 associates with Tax/IKK signalosomes in peri-golgian structures. ( a , b ) Jurkat cells transiently expressing Tax-His and HTLV-1 chronically infected C91PL cells were analyzed by confocal microscopy after staining with His- or Tax- (green), p62- (red), and GM130- ( a , white) and IKKγ-specific ( b , white) antibodies. Nuclei were counterstained with DAPI (blue). Representative images are shown. Tax, p62, GM130 and IKKγ signals were quantified along the segments represented on the merge panels and plotted on the histogram. Scale bar = 10 μm. ( c ) Lysates from HeLa cells transiently expressing Tax-His and/or IKKγ-FLAG were submitted to a FLAG-immunoprecipitation followed by a His-specific Ni-NTA purification and western blot analyses. Full-length blots are presented in Supplementary Fig. S4 .

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).

    Techniques: Expressing, Infection, Confocal Microscopy, Staining, Immunoprecipitation, Purification, Western Blot

    p62 directly interacts with Tax through its 170-206 domain. ( a ) GST and GST-tagged p62 were expressed in bacteria and used for GST pulldown of in vitro translated 35 S-labeled Tax. Inputs as well as eluates were run on SDS-PAGE gels and autoradiography was performed. ( b ) Domain organization of p62 constructs with different deletion used for GST and MBP pulldown assays. The results of the pulldown assays shown in ( c , d ) indicate Tax binding ability and are indicated on the right. PB1, Phox and Bem1 domain; ZZ, zinc finger domain; MIR, multiple protein interaction region; LIR, LC3-interacting region; KIR, KEAP1 interacting region; UBA, ubiquitin-associated domain. ( c , d ) GST pulldown ( c ) and MBP pulldown assays ( d ) were performed with different p62 constructs. The percentage of input radioactively labeled-Tax bound to p62 was quantified from three independent experiments. CBB, Coomassie brilliant blue-stained SDS-PAGE gel.

    Journal: Scientific Reports

    Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function

    doi: 10.1038/s41598-019-52408-x

    Figure Lengend Snippet: p62 directly interacts with Tax through its 170-206 domain. ( a ) GST and GST-tagged p62 were expressed in bacteria and used for GST pulldown of in vitro translated 35 S-labeled Tax. Inputs as well as eluates were run on SDS-PAGE gels and autoradiography was performed. ( b ) Domain organization of p62 constructs with different deletion used for GST and MBP pulldown assays. The results of the pulldown assays shown in ( c , d ) indicate Tax binding ability and are indicated on the right. PB1, Phox and Bem1 domain; ZZ, zinc finger domain; MIR, multiple protein interaction region; LIR, LC3-interacting region; KIR, KEAP1 interacting region; UBA, ubiquitin-associated domain. ( c , d ) GST pulldown ( c ) and MBP pulldown assays ( d ) were performed with different p62 constructs. The percentage of input radioactively labeled-Tax bound to p62 was quantified from three independent experiments. CBB, Coomassie brilliant blue-stained SDS-PAGE gel.

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).

    Techniques: In Vitro, Labeling, SDS Page, Autoradiography, Construct, Binding Assay, Staining

    Functional validation of the BirA*-Tax fusion protein and identification of p62 as a new candidate partner of Tax. ( a ) Lysates from HEK293T cells transfected with the indicated plasmids for 24 h and then treated overnight with biotin or left untreated were analyzed by western blot. ( b ) U2OS cells transiently expressing Tax-His or Myc-BirA*-Tax-His and treated overnight with biotin were analyzed by epifluorescence microscopy after staining with Streptavidin (Strept., green) and His-specific antibodies (red). Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar = 10 μm. The arrows indicate perinuclear accumulation of Tax reminiscent of the Tax/IKK signalosome. ( c ) HEK293T cells were transfected with Myc-BirA* or Myc-BirA*-Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the “Myc-BirA*” condition. The graph shows the result from a representative experiment. ( d ) Lysates from HEK293T cells transiently expressing Myc-BirA* or Myc-BirA*-Tax-His were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. WCL, whole cell lysate. ( e ) SQSTM-1/p62 is a BirA*-Tax-specific biotinylated protein identified by mass spectrometry. ( f ) Lysates from HTLV-1 chronically infected cells (C8166, HuT102 or C91PL cells) were immunoprecipitated with a p62-specific or Tax-specific antibody, or with control Ig (IP Ig). Samples were then analyzed by western blot. WCL, whole cell lysates. Full-length blots are presented in Supplementary Fig. S4 .

    Journal: Scientific Reports

    Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function

    doi: 10.1038/s41598-019-52408-x

    Figure Lengend Snippet: Functional validation of the BirA*-Tax fusion protein and identification of p62 as a new candidate partner of Tax. ( a ) Lysates from HEK293T cells transfected with the indicated plasmids for 24 h and then treated overnight with biotin or left untreated were analyzed by western blot. ( b ) U2OS cells transiently expressing Tax-His or Myc-BirA*-Tax-His and treated overnight with biotin were analyzed by epifluorescence microscopy after staining with Streptavidin (Strept., green) and His-specific antibodies (red). Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bar = 10 μm. The arrows indicate perinuclear accumulation of Tax reminiscent of the Tax/IKK signalosome. ( c ) HEK293T cells were transfected with Myc-BirA* or Myc-BirA*-Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the “Myc-BirA*” condition. The graph shows the result from a representative experiment. ( d ) Lysates from HEK293T cells transiently expressing Myc-BirA* or Myc-BirA*-Tax-His were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. WCL, whole cell lysate. ( e ) SQSTM-1/p62 is a BirA*-Tax-specific biotinylated protein identified by mass spectrometry. ( f ) Lysates from HTLV-1 chronically infected cells (C8166, HuT102 or C91PL cells) were immunoprecipitated with a p62-specific or Tax-specific antibody, or with control Ig (IP Ig). Samples were then analyzed by western blot. WCL, whole cell lysates. Full-length blots are presented in Supplementary Fig. S4 .

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).

    Techniques: Functional Assay, Transfection, Western Blot, Expressing, Epifluorescence Microscopy, Staining, Construct, Luciferase, Activity Assay, Mass Spectrometry, Infection, Immunoprecipitation

    p62 binding to ubiquitin is required for p62 potentiation of Tax-mediated NF-κB activation. ( a ) Jurkat cells were transfected with full-length Myc-p62 (My-p62 FL) or p62 mutants in which the Tax-interacting region (Myc-p62 Δ170-221) or the ubiquitin-binding domain (Myc-p62 ΔUBA) were deleted, and an NF-κB-luc construct, followed by transduction with an empty or Flag-Tax-encoding lentivector. Luciferase activity was measured and normalized to the corresponding Tax-negative condition. Values obtained with full-length ectopic p62 were set to 1 and other values are shown as fold change over the “p62 FL” condition. The graph shows results from 3 independent experiments. ( b ) Lysates from p62 −/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 Δ170-221 were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. ( c ) Lysates from p62 −/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 ΔUBA were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. ( d ) HeLa cells were transfected with control (−) or p62 -specific (+) siRNA and Tax-His. Cell lysates were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. ( e ) Lysates from WT and p62 −/− MEF cells transiently expressing Tax-His- and FLAG-IKKγ were immunoprecipitated with a FLAG-specific antibody followed by western blot analyses. **p

    Journal: Scientific Reports

    Article Title: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function

    doi: 10.1038/s41598-019-52408-x

    Figure Lengend Snippet: p62 binding to ubiquitin is required for p62 potentiation of Tax-mediated NF-κB activation. ( a ) Jurkat cells were transfected with full-length Myc-p62 (My-p62 FL) or p62 mutants in which the Tax-interacting region (Myc-p62 Δ170-221) or the ubiquitin-binding domain (Myc-p62 ΔUBA) were deleted, and an NF-κB-luc construct, followed by transduction with an empty or Flag-Tax-encoding lentivector. Luciferase activity was measured and normalized to the corresponding Tax-negative condition. Values obtained with full-length ectopic p62 were set to 1 and other values are shown as fold change over the “p62 FL” condition. The graph shows results from 3 independent experiments. ( b ) Lysates from p62 −/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 Δ170-221 were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. ( c ) Lysates from p62 −/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 ΔUBA were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. ( d ) HeLa cells were transfected with control (−) or p62 -specific (+) siRNA and Tax-His. Cell lysates were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. ( e ) Lysates from WT and p62 −/− MEF cells transiently expressing Tax-His- and FLAG-IKKγ were immunoprecipitated with a FLAG-specific antibody followed by western blot analyses. **p

    Article Snippet: Antibodies and reagents The following antibodies were used: anti-His (ab9136 or ab18184, Abcam or sc-804, Santa Cruz), anti-Tax1 (Tab172, NIH), anti-p62 (GP62-C, Progen), anti-OPTN (100000, Cayman), anti-GM130 (610823, BD), anti-IKKγ (611306, BD), anti-phosphorylated IKKα/β (2078, Cell Signaling Technology), anti-IκBα (4814, Cell Signaling Technology), anti-phosphorylated IκBα (9246, Cell Signaling Technology), anti-FLAG (M2, Sigma-Aldrich), anti-Myc (4A6, Millipore), anti-Ub (P4D1, sc-8017, Santa Cruz), anti-actin (AC-74, Sigma-Aldrich), anti-Nup153 (147050002, Covance).

    Techniques: Binding Assay, Activation Assay, Transfection, Construct, Transduction, Luciferase, Activity Assay, Expressing, Immunoprecipitation, Western Blot

    Inhibition of autophagy with CQ sensitizes mesothelioma cell lines to PI3K/mTOR inhibitors. ( a ) Anti-P62, L-C3BI/II and Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated with 20 μ M CQ, 0.5 μ M GDC-0980 or in combination for 24, 48 and 72 h. ( b ) Light micrographs of SPC212 and Mero-82 treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Scale bar represents 25 μ m. ( c ) Quantification of the percentage of ATP content of SPC212 cells and Mero-82 cell treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Data are presented as means±S.D. from ≥3 independent experiments. Significance was determined by analysis of variance test (*** P

    Journal: Cell Death & Disease

    Article Title: Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors

    doi: 10.1038/cddis.2015.124

    Figure Lengend Snippet: Inhibition of autophagy with CQ sensitizes mesothelioma cell lines to PI3K/mTOR inhibitors. ( a ) Anti-P62, L-C3BI/II and Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated with 20 μ M CQ, 0.5 μ M GDC-0980 or in combination for 24, 48 and 72 h. ( b ) Light micrographs of SPC212 and Mero-82 treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Scale bar represents 25 μ m. ( c ) Quantification of the percentage of ATP content of SPC212 cells and Mero-82 cell treated with 0.2 μ M NVP-BEZ235, 0.5 μ M GDC-0980 alone or in combination with 20 μ M CQ for 96 h. Data are presented as means±S.D. from ≥3 independent experiments. Significance was determined by analysis of variance test (*** P

    Article Snippet: For western blotting, membranes were probed with the following primary antibodies: rabbit anti-AKT (no. 9272), rabbit anti-Phospho-AKT (Thr308) (no. 9275), rabbit anti-Phospho-AKT (Ser473) (193H12, no. 9275), mouse anti-S6 (54D2, no. 2317), rabbit anti-Phospho-S6 (Ser235/236) (D57.2.2E, no. 4858), rabbit anti-4E-BP1 (no. 9452), rabbit-Phospho-4E-BP1 (Thr37/46) (236B4, no. 2855), rabbit anti-p44/42 MAP Kinase (no. 9102), rabbit anti-Phospho-p44/42 MAP Kinase (Thr202/Tyr 204) (no. 9101), rabbit anti-PTEN (138G6, no. 9559), rabbit anti-LC3B (no. 2775), rabbit anti-PARP (no. 9542), rabbit anti-caspase-3 (no. 9662) from Cell Signaling Technology; rabbit anti-NF2 (C18, no. sc-332) from Santa Cruz Biotechnologies (Dallas, TX, USA); guinea pig anti-p62 (GP62-C) from Progen (Heidelberg, Germany); mouse anti-RIP (no. 610458) and mouse anti-XIAP (no. 610763) from Transduction Laboratories (Allschwil, Switzerland); mouse anti-ATG5 (7C6) from Nanotools (München, Switzerland); rabbit anti-Phospho-NF2 (Ser518) (PAI-14252) from Pierce (Rockford, IL, USA) and mouse anti-Actin (no. 69100) from MP Biomedicals (Santa Ana, CA, USA).

    Techniques: Inhibition, Western Blot

    Autophagy is necessary for mesothelioma cell growth. SPC212 and Mero-82 cell lines were stable transduced with virus particles carrying Mir30- Sh-Atg5 or Mir30-Sh-Ctrl. ( a ) Atg5 gene silencing was confirmed by anti-ATG5 western blotting. ( b ) Anti-P62, -LC3BI/II and -Actin of SPC212 and Mero-82 Sh-Atg5 and Sh-Ctrl untreated or treated with 20 μ M CQ for 4 h. ( c ) Fold cell growth of SPC212 and Mero-82 Sh-Atg5 and Sh-Ctrl for 3 days. Data are presented as means±S.D. from three independent experiments. Significance was determined by Mann–Whitney U -test (* P

    Journal: Cell Death & Disease

    Article Title: Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors

    doi: 10.1038/cddis.2015.124

    Figure Lengend Snippet: Autophagy is necessary for mesothelioma cell growth. SPC212 and Mero-82 cell lines were stable transduced with virus particles carrying Mir30- Sh-Atg5 or Mir30-Sh-Ctrl. ( a ) Atg5 gene silencing was confirmed by anti-ATG5 western blotting. ( b ) Anti-P62, -LC3BI/II and -Actin of SPC212 and Mero-82 Sh-Atg5 and Sh-Ctrl untreated or treated with 20 μ M CQ for 4 h. ( c ) Fold cell growth of SPC212 and Mero-82 Sh-Atg5 and Sh-Ctrl for 3 days. Data are presented as means±S.D. from three independent experiments. Significance was determined by Mann–Whitney U -test (* P

    Article Snippet: For western blotting, membranes were probed with the following primary antibodies: rabbit anti-AKT (no. 9272), rabbit anti-Phospho-AKT (Thr308) (no. 9275), rabbit anti-Phospho-AKT (Ser473) (193H12, no. 9275), mouse anti-S6 (54D2, no. 2317), rabbit anti-Phospho-S6 (Ser235/236) (D57.2.2E, no. 4858), rabbit anti-4E-BP1 (no. 9452), rabbit-Phospho-4E-BP1 (Thr37/46) (236B4, no. 2855), rabbit anti-p44/42 MAP Kinase (no. 9102), rabbit anti-Phospho-p44/42 MAP Kinase (Thr202/Tyr 204) (no. 9101), rabbit anti-PTEN (138G6, no. 9559), rabbit anti-LC3B (no. 2775), rabbit anti-PARP (no. 9542), rabbit anti-caspase-3 (no. 9662) from Cell Signaling Technology; rabbit anti-NF2 (C18, no. sc-332) from Santa Cruz Biotechnologies (Dallas, TX, USA); guinea pig anti-p62 (GP62-C) from Progen (Heidelberg, Germany); mouse anti-RIP (no. 610458) and mouse anti-XIAP (no. 610763) from Transduction Laboratories (Allschwil, Switzerland); mouse anti-ATG5 (7C6) from Nanotools (München, Switzerland); rabbit anti-Phospho-NF2 (Ser518) (PAI-14252) from Pierce (Rockford, IL, USA) and mouse anti-Actin (no. 69100) from MP Biomedicals (Santa Ana, CA, USA).

    Techniques: Transduction, Western Blot, MANN-WHITNEY

    Mesothelioma cells exhibit a high level of autophagy, which is increased upon inhibition of PI3K/mTOR signaling. ( a ) Anti-P62 and -LC3BI/II and -Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated for 72 h with increasing concentrations of NVP-BEZ235 or GDC-0980 as indicated. ( b ) Anti-LC3BI/II and -Actin western blots of mesothelial cell line SDM104 and MPM cell lines SPC111, SPC212, NCI-H226, NCI-H2052, NCI-H2452, Mero-14, Mero-25, Mero-82, Mero-83, Mero-84, Mero-95, ACC-Meso-1, ACC-Meso-4, MSTO-211H and ONE58 left with serum or serum-starved for 16 h. In the lower panel, protein level quantification of LC3BII normalized against Actin is represented for the cell lines described above with standard culture conditions

    Journal: Cell Death & Disease

    Article Title: Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors

    doi: 10.1038/cddis.2015.124

    Figure Lengend Snippet: Mesothelioma cells exhibit a high level of autophagy, which is increased upon inhibition of PI3K/mTOR signaling. ( a ) Anti-P62 and -LC3BI/II and -Actin western blots of protein lysates of sensitive cell line SPC212 and resistant cell line Mero-82 treated for 72 h with increasing concentrations of NVP-BEZ235 or GDC-0980 as indicated. ( b ) Anti-LC3BI/II and -Actin western blots of mesothelial cell line SDM104 and MPM cell lines SPC111, SPC212, NCI-H226, NCI-H2052, NCI-H2452, Mero-14, Mero-25, Mero-82, Mero-83, Mero-84, Mero-95, ACC-Meso-1, ACC-Meso-4, MSTO-211H and ONE58 left with serum or serum-starved for 16 h. In the lower panel, protein level quantification of LC3BII normalized against Actin is represented for the cell lines described above with standard culture conditions

    Article Snippet: For western blotting, membranes were probed with the following primary antibodies: rabbit anti-AKT (no. 9272), rabbit anti-Phospho-AKT (Thr308) (no. 9275), rabbit anti-Phospho-AKT (Ser473) (193H12, no. 9275), mouse anti-S6 (54D2, no. 2317), rabbit anti-Phospho-S6 (Ser235/236) (D57.2.2E, no. 4858), rabbit anti-4E-BP1 (no. 9452), rabbit-Phospho-4E-BP1 (Thr37/46) (236B4, no. 2855), rabbit anti-p44/42 MAP Kinase (no. 9102), rabbit anti-Phospho-p44/42 MAP Kinase (Thr202/Tyr 204) (no. 9101), rabbit anti-PTEN (138G6, no. 9559), rabbit anti-LC3B (no. 2775), rabbit anti-PARP (no. 9542), rabbit anti-caspase-3 (no. 9662) from Cell Signaling Technology; rabbit anti-NF2 (C18, no. sc-332) from Santa Cruz Biotechnologies (Dallas, TX, USA); guinea pig anti-p62 (GP62-C) from Progen (Heidelberg, Germany); mouse anti-RIP (no. 610458) and mouse anti-XIAP (no. 610763) from Transduction Laboratories (Allschwil, Switzerland); mouse anti-ATG5 (7C6) from Nanotools (München, Switzerland); rabbit anti-Phospho-NF2 (Ser518) (PAI-14252) from Pierce (Rockford, IL, USA) and mouse anti-Actin (no. 69100) from MP Biomedicals (Santa Ana, CA, USA).

    Techniques: Inhibition, Western Blot

    The NBD of NOD2 interacts with both TRAF6 and UBA domains of p62. A . NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. B . HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). C . Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

    doi: 10.1371/journal.pone.0057138

    Figure Lengend Snippet: The NBD of NOD2 interacts with both TRAF6 and UBA domains of p62. A . NOD2 (left top panel) and p62 (right top panel) structures, and their mutant constructs are schematically presented. B . HEK293T cells were transfected with GFP-p62 and Myc-NBD (left middle panel), GFP-p62 and Myc-CARD (center middle panel) or GFP-p62 and Myc-ΔLRR (LRR region-deleted NOD2) (right middle panel). C . Similarly, HEK293T cells were transiently transfected with GFP-TRAF6 domain of p62 and Myc-ΔLRR (left bottom panel), GFP-UBA domain of p62 and Myc-ΔLRR (middle bottom panel), and GFP-PB1 domain of p62 and Myc-ΔLRR (right bottom panel); co-immunoprecipitation assays were performed as described in legend to Fig. 2. Data shown are representative images of 3 independent experiments.

    Article Snippet: Blots were probed with primary antibodies including anti-human NOD2 , anti-GFP (Clontech), anti-Myc (Clontech), anti-HA (Abcam), anti-LC3 (Cell Signaling Technology), anti-p62 (Abnova), anti-phospho p38 (Cell Signaling Technology), anti-NOD2 4A11 , and anti-p38 (loading control).

    Techniques: Mutagenesis, Construct, Transfection, Immunoprecipitation

    p62 stabilizes gMDP-induced NOD2 oligomers. A . HEK293T cells were stably transfected with pLNCX-NOD2 as described in “Methods”. These cells were treated with scramble (si-Scramble) or p62 targeting (si-p62) small interference RNAs for 24 h. Cells were then treated with the translation inhibitor cyclohexamide (CHX, 100 µg/ml) and gMDP (5 µg/ml) for the time indicated, and immunoblots against NOD2 were performed. Intensities of NOD2 bands in comparison with p38 bands (loading control) were expressed as 100% for control samples (right panel). The ImageJ (NIH) program was used for densitometry analysis and data were expressed as mean ± S.D. (n≥4). *p

    Journal: PLoS ONE

    Article Title: p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

    doi: 10.1371/journal.pone.0057138

    Figure Lengend Snippet: p62 stabilizes gMDP-induced NOD2 oligomers. A . HEK293T cells were stably transfected with pLNCX-NOD2 as described in “Methods”. These cells were treated with scramble (si-Scramble) or p62 targeting (si-p62) small interference RNAs for 24 h. Cells were then treated with the translation inhibitor cyclohexamide (CHX, 100 µg/ml) and gMDP (5 µg/ml) for the time indicated, and immunoblots against NOD2 were performed. Intensities of NOD2 bands in comparison with p38 bands (loading control) were expressed as 100% for control samples (right panel). The ImageJ (NIH) program was used for densitometry analysis and data were expressed as mean ± S.D. (n≥4). *p

    Article Snippet: Blots were probed with primary antibodies including anti-human NOD2 , anti-GFP (Clontech), anti-Myc (Clontech), anti-HA (Abcam), anti-LC3 (Cell Signaling Technology), anti-p62 (Abnova), anti-phospho p38 (Cell Signaling Technology), anti-NOD2 4A11 , and anti-p38 (loading control).

    Techniques: Stable Transfection, Transfection, Western Blot

    p62 co-localizes with NOD2 through the NBD domain of NOD2. A . HEK293T cells were transfected for 24 h with scramble siRNA (left top panel), si-p62 (right top panel), and GFP-NOD2. GFP-NOD2 was visualized using confocal microscopy as described in “Methods”. B . Similarly, DsRed-NBD domain, LRR region or full-length NOD2 and GFP-p62 expression vectors were transfected in HEK293T cells and co-localization of these proteins was examined using confocal microscopy. C . Immunogold staining of co-localized pCMV-HA-p62 (18 nm colloidal gold) and GFP-NOD2 (10 nm colloidal gold) in HEK293T cells. Cells on grids were viewed using a transmission electron microscope. Scale bars: 500 nm (left bottom), 100 nm (middle, right bottom). D . HEK293T cells were transfected with DsRed-NOD2 and GFP-LC3 plasmids. Cells were observed by confocal microscopy and images were acquired using ZEN software.

    Journal: PLoS ONE

    Article Title: p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

    doi: 10.1371/journal.pone.0057138

    Figure Lengend Snippet: p62 co-localizes with NOD2 through the NBD domain of NOD2. A . HEK293T cells were transfected for 24 h with scramble siRNA (left top panel), si-p62 (right top panel), and GFP-NOD2. GFP-NOD2 was visualized using confocal microscopy as described in “Methods”. B . Similarly, DsRed-NBD domain, LRR region or full-length NOD2 and GFP-p62 expression vectors were transfected in HEK293T cells and co-localization of these proteins was examined using confocal microscopy. C . Immunogold staining of co-localized pCMV-HA-p62 (18 nm colloidal gold) and GFP-NOD2 (10 nm colloidal gold) in HEK293T cells. Cells on grids were viewed using a transmission electron microscope. Scale bars: 500 nm (left bottom), 100 nm (middle, right bottom). D . HEK293T cells were transfected with DsRed-NOD2 and GFP-LC3 plasmids. Cells were observed by confocal microscopy and images were acquired using ZEN software.

    Article Snippet: Blots were probed with primary antibodies including anti-human NOD2 , anti-GFP (Clontech), anti-Myc (Clontech), anti-HA (Abcam), anti-LC3 (Cell Signaling Technology), anti-p62 (Abnova), anti-phospho p38 (Cell Signaling Technology), anti-NOD2 4A11 , and anti-p38 (loading control).

    Techniques: Transfection, Confocal Microscopy, Expressing, Staining, Transmission Assay, Microscopy, Software

    NOD2 physically interacts with p62. A–C . HEK293T cells were transiently transfected with expression vectors encoding GFP-tagged p62 (GFP-p62) and/or Myc-tagged NOD2 (Myc-NOD2). After 24 h, total cell lysates were subjected to immunoprecipitation using anti-GFP (A) or anti-Myc (B) antibodies and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against GFP and HA. C . Similar experiments as A-B were performed but with or without N-glycorylated muramyldipeptide (gMDP: 5 µg/mL) treatments for 4 h. Data shown are representative images of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

    doi: 10.1371/journal.pone.0057138

    Figure Lengend Snippet: NOD2 physically interacts with p62. A–C . HEK293T cells were transiently transfected with expression vectors encoding GFP-tagged p62 (GFP-p62) and/or Myc-tagged NOD2 (Myc-NOD2). After 24 h, total cell lysates were subjected to immunoprecipitation using anti-GFP (A) or anti-Myc (B) antibodies and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against GFP and HA. C . Similar experiments as A-B were performed but with or without N-glycorylated muramyldipeptide (gMDP: 5 µg/mL) treatments for 4 h. Data shown are representative images of 3 independent experiments.

    Article Snippet: Blots were probed with primary antibodies including anti-human NOD2 , anti-GFP (Clontech), anti-Myc (Clontech), anti-HA (Abcam), anti-LC3 (Cell Signaling Technology), anti-p62 (Abnova), anti-phospho p38 (Cell Signaling Technology), anti-NOD2 4A11 , and anti-p38 (loading control).

    Techniques: Transfection, Expressing, Immunoprecipitation, SDS Page

    p62 enhances pro-IL-1β expression and TNF-α production in macrophages. A . RAW 264.7 cells were transfected with scrambled (si-Scramble) or p62 targeting (si-p62) small interference RNAs using Lipofectamine™ 2000. Twenty four hours post-transfection, cells were treated with a low dose of LPS (50 ng/mL) for 4 h, rinsed with complete media twice, and then incubated with gMDP (5 µg/mL) for another 4 h. Expression of pro-IL-1β was detected using an antibody against IL-1β and p38 was used as a loading control. Densitometric analysis of blots was done using ImageJ (NIH). Data are expressed as mean ± S.D. (n = 3). N.S., not significant; * p

    Journal: PLoS ONE

    Article Title: p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

    doi: 10.1371/journal.pone.0057138

    Figure Lengend Snippet: p62 enhances pro-IL-1β expression and TNF-α production in macrophages. A . RAW 264.7 cells were transfected with scrambled (si-Scramble) or p62 targeting (si-p62) small interference RNAs using Lipofectamine™ 2000. Twenty four hours post-transfection, cells were treated with a low dose of LPS (50 ng/mL) for 4 h, rinsed with complete media twice, and then incubated with gMDP (5 µg/mL) for another 4 h. Expression of pro-IL-1β was detected using an antibody against IL-1β and p38 was used as a loading control. Densitometric analysis of blots was done using ImageJ (NIH). Data are expressed as mean ± S.D. (n = 3). N.S., not significant; * p

    Article Snippet: Blots were probed with primary antibodies including anti-human NOD2 , anti-GFP (Clontech), anti-Myc (Clontech), anti-HA (Abcam), anti-LC3 (Cell Signaling Technology), anti-p62 (Abnova), anti-phospho p38 (Cell Signaling Technology), anti-NOD2 4A11 , and anti-p38 (loading control).

    Techniques: Expressing, Transfection, Incubation

    p62 is required for the activation of NF-κB and p38 MAPK, and ubiquitination of RIP2 and TRAF6. A . HEK293T cells were transfected with pCMV-Myc-NOD2 and NF-κB luciferase reporter constructs in the presence of scrambled or p62-targeting small interference RNAs (si-p62). Cells were then treated with gMDP (5 µg/mL) for 4 h and NF-κB activity was measured. Data are expressed as the fold of luciferase activity ± SD (n = 3). B-C . HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then transfected with expression vectors for HA-ubiquitin (HA-Ub) and pcDNA3-Myc-RIP2 or pCMV-Myc-TRAF6 for another 24 h. After treating the cells with gMDP (5 µg/mL) for 4 h, RIP2 (B) or TRAF6 (C) was immunoprecipitated with Myc antibodies from total cell lysates and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against HA. Myc-RIP2 or Myc-TRAF6, NOD2 and HA-ubiquitin were analyzed by immunoblot as the inputs (bottom panels). p62 protein levels were also measured by immunoblot. Data shown are representative images of 3 independent experiments. D , HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then treated with gMDP (5 µg/ml) for the times indicated. Activation of p38 was analysed through immunoblotting against tyrosine phosphoryled p38. The ImageJ (NIH) program was used for densitometry analysis of phosphor-p83 bands and data were expressed as mean ± S.D. (n = 3).

    Journal: PLoS ONE

    Article Title: p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization

    doi: 10.1371/journal.pone.0057138

    Figure Lengend Snippet: p62 is required for the activation of NF-κB and p38 MAPK, and ubiquitination of RIP2 and TRAF6. A . HEK293T cells were transfected with pCMV-Myc-NOD2 and NF-κB luciferase reporter constructs in the presence of scrambled or p62-targeting small interference RNAs (si-p62). Cells were then treated with gMDP (5 µg/mL) for 4 h and NF-κB activity was measured. Data are expressed as the fold of luciferase activity ± SD (n = 3). B-C . HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then transfected with expression vectors for HA-ubiquitin (HA-Ub) and pcDNA3-Myc-RIP2 or pCMV-Myc-TRAF6 for another 24 h. After treating the cells with gMDP (5 µg/mL) for 4 h, RIP2 (B) or TRAF6 (C) was immunoprecipitated with Myc antibodies from total cell lysates and the immune complexes were resolved by SDS-PAGE followed by immunoblotting against HA. Myc-RIP2 or Myc-TRAF6, NOD2 and HA-ubiquitin were analyzed by immunoblot as the inputs (bottom panels). p62 protein levels were also measured by immunoblot. Data shown are representative images of 3 independent experiments. D , HEK293T cells stably expressing NOD2 were first treated with scramble siRNA or si-p62 for 24 h, and then treated with gMDP (5 µg/ml) for the times indicated. Activation of p38 was analysed through immunoblotting against tyrosine phosphoryled p38. The ImageJ (NIH) program was used for densitometry analysis of phosphor-p83 bands and data were expressed as mean ± S.D. (n = 3).

    Article Snippet: Blots were probed with primary antibodies including anti-human NOD2 , anti-GFP (Clontech), anti-Myc (Clontech), anti-HA (Abcam), anti-LC3 (Cell Signaling Technology), anti-p62 (Abnova), anti-phospho p38 (Cell Signaling Technology), anti-NOD2 4A11 , and anti-p38 (loading control).

    Techniques: Activation Assay, Transfection, Luciferase, Construct, Activity Assay, Stable Transfection, Expressing, Immunoprecipitation, SDS Page

    More B. cepacia vacuoles colocalize with p62 in WT macrophages than in ΔF508 macrophages. A , confocal microscopy for WT and ΔF508 macrophages infected with B. cepacia -expressing m-RFP for 0.5 or 1.5 h. p62 stained green , whereas nuclei

    Journal: The Journal of Biological Chemistry

    Article Title: Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ?F508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery *

    doi: 10.1074/jbc.M112.411728

    Figure Lengend Snippet: More B. cepacia vacuoles colocalize with p62 in WT macrophages than in ΔF508 macrophages. A , confocal microscopy for WT and ΔF508 macrophages infected with B. cepacia -expressing m-RFP for 0.5 or 1.5 h. p62 stained green , whereas nuclei

    Article Snippet: Rabbit anti-LC3 (Abgent, catalog no. AP1805a), mouse anti-p62 (BD Biosciences, catalog no. 610832), FK2 mAb (Enzo Bioscience, catalog no. BML-PW8810), and rabbit anti-BECN1 (Abcam, catalog no. ab55878) were used, followed by fluorescent secondary antibodies (Molecular Probes, catalog no. ).

    Techniques: Confocal Microscopy, Infection, Expressing, Staining

    p62 overexpression decreases growth of B. cepacia in WT macrophages, whereas increases bacterial growth in ΔF508 macrophages. A and D , WT and ΔF508 macrophages were nucleofected with the p62 plasmid or vector control 24 h prior to infection

    Journal: The Journal of Biological Chemistry

    Article Title: Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ?F508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery *

    doi: 10.1074/jbc.M112.411728

    Figure Lengend Snippet: p62 overexpression decreases growth of B. cepacia in WT macrophages, whereas increases bacterial growth in ΔF508 macrophages. A and D , WT and ΔF508 macrophages were nucleofected with the p62 plasmid or vector control 24 h prior to infection

    Article Snippet: Rabbit anti-LC3 (Abgent, catalog no. AP1805a), mouse anti-p62 (BD Biosciences, catalog no. 610832), FK2 mAb (Enzo Bioscience, catalog no. BML-PW8810), and rabbit anti-BECN1 (Abcam, catalog no. ab55878) were used, followed by fluorescent secondary antibodies (Molecular Probes, catalog no. ).

    Techniques: Over Expression, Plasmid Preparation, Infection

    Down-regulation of p62 results in increased growth of B. cepacia in WT murine macrophages, whereas in ΔF508 macrophages it leads to decreased growth. A and D , WT and ΔF508 macrophages were nucleofected with siRNA against p62 ( siRNA-p62

    Journal: The Journal of Biological Chemistry

    Article Title: Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ?F508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery *

    doi: 10.1074/jbc.M112.411728

    Figure Lengend Snippet: Down-regulation of p62 results in increased growth of B. cepacia in WT murine macrophages, whereas in ΔF508 macrophages it leads to decreased growth. A and D , WT and ΔF508 macrophages were nucleofected with siRNA against p62 ( siRNA-p62

    Article Snippet: Rabbit anti-LC3 (Abgent, catalog no. AP1805a), mouse anti-p62 (BD Biosciences, catalog no. 610832), FK2 mAb (Enzo Bioscience, catalog no. BML-PW8810), and rabbit anti-BECN1 (Abcam, catalog no. ab55878) were used, followed by fluorescent secondary antibodies (Molecular Probes, catalog no. ).

    Techniques:

    Colocalization of B. cepacia with BECN1 is increased in ΔF508 macrophages upon depletion of p62. A , confocal microscopy for WT and ΔF508 macrophages infected with B. cepacia -expressing m-RFP for 2 h. BECN1 stained green , and nuclei were

    Journal: The Journal of Biological Chemistry

    Article Title: Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ?F508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery *

    doi: 10.1074/jbc.M112.411728

    Figure Lengend Snippet: Colocalization of B. cepacia with BECN1 is increased in ΔF508 macrophages upon depletion of p62. A , confocal microscopy for WT and ΔF508 macrophages infected with B. cepacia -expressing m-RFP for 2 h. BECN1 stained green , and nuclei were

    Article Snippet: Rabbit anti-LC3 (Abgent, catalog no. AP1805a), mouse anti-p62 (BD Biosciences, catalog no. 610832), FK2 mAb (Enzo Bioscience, catalog no. BML-PW8810), and rabbit anti-BECN1 (Abcam, catalog no. ab55878) were used, followed by fluorescent secondary antibodies (Molecular Probes, catalog no. ).

    Techniques: Confocal Microscopy, Infection, Expressing, Staining

    Depletion of p62 in ΔF508 macrophages improves clearance of B. cepacia by autophagosomes via NDP52 and NBR1. A , B , and C , WT ( A ) and ΔF508 ( B and C ) macrophages were nucleofected with siRNA against NDP52 ( si-NDP52 ), NBR1 ( si-NBR1 ), or

    Journal: The Journal of Biological Chemistry

    Article Title: Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ?F508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery *

    doi: 10.1074/jbc.M112.411728

    Figure Lengend Snippet: Depletion of p62 in ΔF508 macrophages improves clearance of B. cepacia by autophagosomes via NDP52 and NBR1. A , B , and C , WT ( A ) and ΔF508 ( B and C ) macrophages were nucleofected with siRNA against NDP52 ( si-NDP52 ), NBR1 ( si-NBR1 ), or

    Article Snippet: Rabbit anti-LC3 (Abgent, catalog no. AP1805a), mouse anti-p62 (BD Biosciences, catalog no. 610832), FK2 mAb (Enzo Bioscience, catalog no. BML-PW8810), and rabbit anti-BECN1 (Abcam, catalog no. ab55878) were used, followed by fluorescent secondary antibodies (Molecular Probes, catalog no. ).

    Techniques:

    Down-regulation of p62 decreases B. cepacia colocalization with LC3 in WT macrophages but increases the colocalization in ΔF508 macrophages. A and B , confocal microscopy for WT macrophages and ΔF508 macrophages infected with B. cepacia

    Journal: The Journal of Biological Chemistry

    Article Title: Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ?F508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery *

    doi: 10.1074/jbc.M112.411728

    Figure Lengend Snippet: Down-regulation of p62 decreases B. cepacia colocalization with LC3 in WT macrophages but increases the colocalization in ΔF508 macrophages. A and B , confocal microscopy for WT macrophages and ΔF508 macrophages infected with B. cepacia

    Article Snippet: Rabbit anti-LC3 (Abgent, catalog no. AP1805a), mouse anti-p62 (BD Biosciences, catalog no. 610832), FK2 mAb (Enzo Bioscience, catalog no. BML-PW8810), and rabbit anti-BECN1 (Abcam, catalog no. ab55878) were used, followed by fluorescent secondary antibodies (Molecular Probes, catalog no. ).

    Techniques: Confocal Microscopy, Infection

    Murine bone marrow-derived macrophages harboring the ΔF508 mutation have a higher level of p62 than WT macrophages. A , upper panel , immunoblot analysis of WT and ΔF508 macrophage lysates showing the expression level of LC3 (I/II) and p62,

    Journal: The Journal of Biological Chemistry

    Article Title: Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ?F508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery *

    doi: 10.1074/jbc.M112.411728

    Figure Lengend Snippet: Murine bone marrow-derived macrophages harboring the ΔF508 mutation have a higher level of p62 than WT macrophages. A , upper panel , immunoblot analysis of WT and ΔF508 macrophage lysates showing the expression level of LC3 (I/II) and p62,

    Article Snippet: Rabbit anti-LC3 (Abgent, catalog no. AP1805a), mouse anti-p62 (BD Biosciences, catalog no. 610832), FK2 mAb (Enzo Bioscience, catalog no. BML-PW8810), and rabbit anti-BECN1 (Abcam, catalog no. ab55878) were used, followed by fluorescent secondary antibodies (Molecular Probes, catalog no. ).

    Techniques: Derivative Assay, Mutagenesis, Expressing

    CALCOCO 1 binds both to LDS and UDS of ATG 8 family proteins GST pull‐down testing binding of indicated in vitro transcribed/translated 35 S‐Myc‐CALCOCO1 constructs with indicated recombinant GST‐tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right). GST pull‐down assays of in vitro transcribed/translated 35 S‐Myc‐CALCOCO1 and 35 S‐Myc‐p62 with recombinant GST‐GABARAPL2 (WT and indicated mutants). GST pull‐down assays of in vitro transcribed/translated 35 S‐Myc‐TAX1BP1 (WT and indicated mutants) with recombinant GST‐GABARAP (WT and indicated mutants). Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP‐CALCOCO1 or EGFP‐CALCOCO1 mLIR + Δ623–691. Cells were induced with tetracycline for 24 h and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one Western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In (F), the bars represent the mean ± SD of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as *** P ˂ 0.001, * P ˂ 0.01; ns is not significant. HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 or EGFP‐CALCOCO1 mLIR + Δ623–691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: CALCOCO1 acts with VAMP‐associated proteins to mediate ER‐phagy

    doi: 10.15252/embj.2019103649

    Figure Lengend Snippet: CALCOCO 1 binds both to LDS and UDS of ATG 8 family proteins GST pull‐down testing binding of indicated in vitro transcribed/translated 35 S‐Myc‐CALCOCO1 constructs with indicated recombinant GST‐tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right). GST pull‐down assays of in vitro transcribed/translated 35 S‐Myc‐CALCOCO1 and 35 S‐Myc‐p62 with recombinant GST‐GABARAPL2 (WT and indicated mutants). GST pull‐down assays of in vitro transcribed/translated 35 S‐Myc‐TAX1BP1 (WT and indicated mutants) with recombinant GST‐GABARAP (WT and indicated mutants). Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP‐CALCOCO1 or EGFP‐CALCOCO1 mLIR + Δ623–691. Cells were induced with tetracycline for 24 h and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one Western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In (F), the bars represent the mean ± SD of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as *** P ˂ 0.001, * P ˂ 0.01; ns is not significant. HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 or EGFP‐CALCOCO1 mLIR + Δ623–691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm. Source data are available online for this figure.

    Article Snippet: AntibodiesMouse monoclonal anti‐CALCOCO1 (A‐10) (Santa Cruz Biotech Cat#sc‐515670), rabbit polyclonal anti‐CALCOCO1 (Sigma‐Aldrich Cat#HPA038314), mouse polyclonal anti‐CALCOCO1 (Abcam Cat# ab167237), rabbit polyclonal anti‐VAPA (Proteintech Cat#15275‐1‐AP), rabbit polyclonal anti‐VAPB (clone 4F6A6) (Proteintech Cat#66191‐1‐IG), rabbit polyclonal anti‐GFP (Abcam Cat #ab290), mouse monoclonal anti‐p62 (BD Biosciences Cat #610833), guinea pig polyclonal anti‐p62 (Progen Cat #GP62‐C), rabbit polyclonal anti‐CALCOCO2 (Sigma‐Aldrich Cat #HPA023195), rabbit anti‐CALCOCO2 (Abcam Cat #ab68588), rabbit monoclonal anti‐ATG7 (Cell Signaling Cat #D12B11), rabbit polyclonal anti‐LC3B (Novus Bio Cat #NB100‐2220), rabbit polyclonal anti‐LC3B (Sigma‐Aldrich Cat # L7543), mouse monoclonal anti‐GABARAP (MBL Cat # M135‐3), mouse monoclonal anti‐Myc tag (9B11) cell signaling #2276), mouse monoclonal anti‐RTN3 (F‐6) (Santa Cruz Biotechnology Cat #sc‐374599), rabbit polyclonal FAM134B (Proteintech Cat #21537‐1‐AP), mouse polyclonal anti‐NBR1 (Santa cruz biotechnology #sc‐130380), rabbit polyclonal anti‐CKAP4(CLIMP63) (Proteintech Cat#16686‐1‐AP), rabbit polyclonal anti‐TEX264 (Novus Bio Cat #NBP1‐89866), rabbit polyclonal anti‐GAPDH (Sigma‐Aldrich Cat#G9545), rabbit polyclonal anti‐actin (Sigma‐Aldrich Cat #A2066), HRP‐conjugated goat polyclonal anti‐rabbit (BD Biosciences Cat #554021), HRP‐conjugated goat polyclonal anti‐mouse (BD Biosciences Cat #554002).

    Techniques: Binding Assay, In Vitro, Construct, Recombinant, Stable Transfection, Transfection, Western Blot, Expressing

    CALCOCO 1 is degraded by macro‐autophagy Domain architecture of CALCOCO paralogs showing the SKICH domain, a conserved LIR motif (LVV), coiled‐coli regions (CC), and zinc finger domains (ZF). Immunoblot analysis of indicated cell lines, starved for 6 h (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In (C, E), endogenous CALCOCO1 is analyzed and the bars represent the mean ± SD of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as *** P ˂ 0.001, ** P ˂ 0.005, * P ˂ 0.01; ns is not significant. A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 and 2 μm (zoomed inset). Same cells as in (E) were left untreated or treated with Baf A1 for 6 h and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans. CALCOCO1, p62, and LC3B puncta in the indicated conditions, counted using an automated system. The error bars represent mean ± SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test. Significance is displayed as *** P

    Journal: The EMBO Journal

    Article Title: CALCOCO1 acts with VAMP‐associated proteins to mediate ER‐phagy

    doi: 10.15252/embj.2019103649

    Figure Lengend Snippet: CALCOCO 1 is degraded by macro‐autophagy Domain architecture of CALCOCO paralogs showing the SKICH domain, a conserved LIR motif (LVV), coiled‐coli regions (CC), and zinc finger domains (ZF). Immunoblot analysis of indicated cell lines, starved for 6 h (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In (C, E), endogenous CALCOCO1 is analyzed and the bars represent the mean ± SD of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test and significance displayed as *** P ˂ 0.001, ** P ˂ 0.005, * P ˂ 0.01; ns is not significant. A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP‐CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 and 2 μm (zoomed inset). Same cells as in (E) were left untreated or treated with Baf A1 for 6 h and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans. CALCOCO1, p62, and LC3B puncta in the indicated conditions, counted using an automated system. The error bars represent mean ± SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one‐way ANOVA followed by Tukey multiple comparison test. Significance is displayed as *** P

    Article Snippet: AntibodiesMouse monoclonal anti‐CALCOCO1 (A‐10) (Santa Cruz Biotech Cat#sc‐515670), rabbit polyclonal anti‐CALCOCO1 (Sigma‐Aldrich Cat#HPA038314), mouse polyclonal anti‐CALCOCO1 (Abcam Cat# ab167237), rabbit polyclonal anti‐VAPA (Proteintech Cat#15275‐1‐AP), rabbit polyclonal anti‐VAPB (clone 4F6A6) (Proteintech Cat#66191‐1‐IG), rabbit polyclonal anti‐GFP (Abcam Cat #ab290), mouse monoclonal anti‐p62 (BD Biosciences Cat #610833), guinea pig polyclonal anti‐p62 (Progen Cat #GP62‐C), rabbit polyclonal anti‐CALCOCO2 (Sigma‐Aldrich Cat #HPA023195), rabbit anti‐CALCOCO2 (Abcam Cat #ab68588), rabbit monoclonal anti‐ATG7 (Cell Signaling Cat #D12B11), rabbit polyclonal anti‐LC3B (Novus Bio Cat #NB100‐2220), rabbit polyclonal anti‐LC3B (Sigma‐Aldrich Cat # L7543), mouse monoclonal anti‐GABARAP (MBL Cat # M135‐3), mouse monoclonal anti‐Myc tag (9B11) cell signaling #2276), mouse monoclonal anti‐RTN3 (F‐6) (Santa Cruz Biotechnology Cat #sc‐374599), rabbit polyclonal FAM134B (Proteintech Cat #21537‐1‐AP), mouse polyclonal anti‐NBR1 (Santa cruz biotechnology #sc‐130380), rabbit polyclonal anti‐CKAP4(CLIMP63) (Proteintech Cat#16686‐1‐AP), rabbit polyclonal anti‐TEX264 (Novus Bio Cat #NBP1‐89866), rabbit polyclonal anti‐GAPDH (Sigma‐Aldrich Cat#G9545), rabbit polyclonal anti‐actin (Sigma‐Aldrich Cat #A2066), HRP‐conjugated goat polyclonal anti‐rabbit (BD Biosciences Cat #554021), HRP‐conjugated goat polyclonal anti‐mouse (BD Biosciences Cat #554002).

    Techniques: Microscopy, Stable Transfection, Expressing, Confocal Microscopy

    CALCOCO 1 is degraded by autophagy and co‐localizes in HBSS ‐treated cells with p62, LC 3B, and GABARAP Immunoblot analysis of indicated cell lines, starved for 6 h (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated. Extension of Fig 1 G. HeLa CALCOCO1 KO cells stably transfected with EGFP‐CALCOCO1 were induced with tetracycline for 24 h and then starved (HBSS) with or without Baf A1 treatment as indicated, before immunostaining for endogenous p62 and LC3B. Scale bars, 5 μm for confocal microscopy images, 2 μm for airyscans. Extension of Fig 1 J. Percentage of LAMP1 rings associated with a CALCOCO1 structure. The error bars represent mean ± SEM of three independent experiments per condition and 200 cells per experiment. HeLa cells stably transfected with EGFP‐CALCOCO1 were treated with tetracycline for 24 h to induce expression of EGFP‐CALCOCO1. Cells were then starved or not and immunostained with anti‐p62, anti‐LC3, and anti‐GABARAP antibodies as indicated. Co‐localization in dots is indicated by circles and supported using line plots shown below the micrographs. Scale bars, 10 μm.

    Journal: The EMBO Journal

    Article Title: CALCOCO1 acts with VAMP‐associated proteins to mediate ER‐phagy

    doi: 10.15252/embj.2019103649

    Figure Lengend Snippet: CALCOCO 1 is degraded by autophagy and co‐localizes in HBSS ‐treated cells with p62, LC 3B, and GABARAP Immunoblot analysis of indicated cell lines, starved for 6 h (HBSS) as indicated, and treated with MG132 or Baf A1 as indicated. Extension of Fig 1 G. HeLa CALCOCO1 KO cells stably transfected with EGFP‐CALCOCO1 were induced with tetracycline for 24 h and then starved (HBSS) with or without Baf A1 treatment as indicated, before immunostaining for endogenous p62 and LC3B. Scale bars, 5 μm for confocal microscopy images, 2 μm for airyscans. Extension of Fig 1 J. Percentage of LAMP1 rings associated with a CALCOCO1 structure. The error bars represent mean ± SEM of three independent experiments per condition and 200 cells per experiment. HeLa cells stably transfected with EGFP‐CALCOCO1 were treated with tetracycline for 24 h to induce expression of EGFP‐CALCOCO1. Cells were then starved or not and immunostained with anti‐p62, anti‐LC3, and anti‐GABARAP antibodies as indicated. Co‐localization in dots is indicated by circles and supported using line plots shown below the micrographs. Scale bars, 10 μm.

    Article Snippet: AntibodiesMouse monoclonal anti‐CALCOCO1 (A‐10) (Santa Cruz Biotech Cat#sc‐515670), rabbit polyclonal anti‐CALCOCO1 (Sigma‐Aldrich Cat#HPA038314), mouse polyclonal anti‐CALCOCO1 (Abcam Cat# ab167237), rabbit polyclonal anti‐VAPA (Proteintech Cat#15275‐1‐AP), rabbit polyclonal anti‐VAPB (clone 4F6A6) (Proteintech Cat#66191‐1‐IG), rabbit polyclonal anti‐GFP (Abcam Cat #ab290), mouse monoclonal anti‐p62 (BD Biosciences Cat #610833), guinea pig polyclonal anti‐p62 (Progen Cat #GP62‐C), rabbit polyclonal anti‐CALCOCO2 (Sigma‐Aldrich Cat #HPA023195), rabbit anti‐CALCOCO2 (Abcam Cat #ab68588), rabbit monoclonal anti‐ATG7 (Cell Signaling Cat #D12B11), rabbit polyclonal anti‐LC3B (Novus Bio Cat #NB100‐2220), rabbit polyclonal anti‐LC3B (Sigma‐Aldrich Cat # L7543), mouse monoclonal anti‐GABARAP (MBL Cat # M135‐3), mouse monoclonal anti‐Myc tag (9B11) cell signaling #2276), mouse monoclonal anti‐RTN3 (F‐6) (Santa Cruz Biotechnology Cat #sc‐374599), rabbit polyclonal FAM134B (Proteintech Cat #21537‐1‐AP), mouse polyclonal anti‐NBR1 (Santa cruz biotechnology #sc‐130380), rabbit polyclonal anti‐CKAP4(CLIMP63) (Proteintech Cat#16686‐1‐AP), rabbit polyclonal anti‐TEX264 (Novus Bio Cat #NBP1‐89866), rabbit polyclonal anti‐GAPDH (Sigma‐Aldrich Cat#G9545), rabbit polyclonal anti‐actin (Sigma‐Aldrich Cat #A2066), HRP‐conjugated goat polyclonal anti‐rabbit (BD Biosciences Cat #554021), HRP‐conjugated goat polyclonal anti‐mouse (BD Biosciences Cat #554002).

    Techniques: Stable Transfection, Transfection, Immunostaining, Confocal Microscopy, Expressing

    p62 deficiency increases the levels of ubiquitinated proteins after methylmercury (MeHg) treatment. Wild-type and p62-defecient mouse embryonic fibroblasts were seeded in 6-cm dishes for treatments. After treatment with 1 µM MeHg for 0–24 h, whole cell lysates were immunoblotted with anti-ubiquitin and p62 antibodies. GAPDH was used as the loading control.

    Journal: Scientific Reports

    Article Title: Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

    doi: 10.1038/s41598-017-17112-8

    Figure Lengend Snippet: p62 deficiency increases the levels of ubiquitinated proteins after methylmercury (MeHg) treatment. Wild-type and p62-defecient mouse embryonic fibroblasts were seeded in 6-cm dishes for treatments. After treatment with 1 µM MeHg for 0–24 h, whole cell lysates were immunoblotted with anti-ubiquitin and p62 antibodies. GAPDH was used as the loading control.

    Article Snippet: The following primary antibodies were used: anti-p62 (Enzo Life Sciences, Farmingdale, NY, USA, PM045, 1:1000), anti-LC3B (Sigma-Aldrich, L7543, 1:1000), anti-ubiquitin (Cell Signaling Technology, #2118, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, #2118, 1:2000), anti-histone H3 (Cell Signaling Technology, #9715, 1:1000), and anti-lamin A/C (Merck Millipore, Temecula, CA, USA, #05-714, 1:1000).

    Techniques:

    Methylmercury (MeHg) increases p62 protein levels in both the cytosol and aggregate fractions of mouse embryonic fibroblasts (MEFs). p62, LC3-I, and LC3-II protein levels in the soluble (Sol) and insoluble (Ins) fractions from wild-type (WT) MEFs. ( A ) Cells were treated with or without 1 µM MeHg for 24 h. GAPDH and lamin A/C were used as loading controls. ( B ) Cells were treated with 1 µM MeHg for 0–24 h. GAPDH and histone H3 were used as loading controls.

    Journal: Scientific Reports

    Article Title: Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

    doi: 10.1038/s41598-017-17112-8

    Figure Lengend Snippet: Methylmercury (MeHg) increases p62 protein levels in both the cytosol and aggregate fractions of mouse embryonic fibroblasts (MEFs). p62, LC3-I, and LC3-II protein levels in the soluble (Sol) and insoluble (Ins) fractions from wild-type (WT) MEFs. ( A ) Cells were treated with or without 1 µM MeHg for 24 h. GAPDH and lamin A/C were used as loading controls. ( B ) Cells were treated with 1 µM MeHg for 0–24 h. GAPDH and histone H3 were used as loading controls.

    Article Snippet: The following primary antibodies were used: anti-p62 (Enzo Life Sciences, Farmingdale, NY, USA, PM045, 1:1000), anti-LC3B (Sigma-Aldrich, L7543, 1:1000), anti-ubiquitin (Cell Signaling Technology, #2118, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, #2118, 1:2000), anti-histone H3 (Cell Signaling Technology, #9715, 1:1000), and anti-lamin A/C (Merck Millipore, Temecula, CA, USA, #05-714, 1:1000).

    Techniques:

    p62 knockout (KO) mouse embryonic fibroblasts (MEFs) are more sensitive to methylmercury (MeHg) than wild-type MEFs. ( A ) Wild-type and p62KO MEFs were treated with 1 µM MeHg for 24 h then stained with Hoechest 33342 and propidium iodide. Images were captured using confocal microscopy. Hoechest 33342 labeled the cell nuclei blue and propidium iodide labeled dead cells red. Scale bars: 100 µm. ( B ) WT and p62KO MEFs were exposed to various concentrations of MeHg for 24 h. Viability was determined using a CCK-8 assay kit. Closed and hatched bars represent for wild-type and p62KO MEFs, respectively. Values are expressed as means ± SEM (n = 3). * p

    Journal: Scientific Reports

    Article Title: Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

    doi: 10.1038/s41598-017-17112-8

    Figure Lengend Snippet: p62 knockout (KO) mouse embryonic fibroblasts (MEFs) are more sensitive to methylmercury (MeHg) than wild-type MEFs. ( A ) Wild-type and p62KO MEFs were treated with 1 µM MeHg for 24 h then stained with Hoechest 33342 and propidium iodide. Images were captured using confocal microscopy. Hoechest 33342 labeled the cell nuclei blue and propidium iodide labeled dead cells red. Scale bars: 100 µm. ( B ) WT and p62KO MEFs were exposed to various concentrations of MeHg for 24 h. Viability was determined using a CCK-8 assay kit. Closed and hatched bars represent for wild-type and p62KO MEFs, respectively. Values are expressed as means ± SEM (n = 3). * p

    Article Snippet: The following primary antibodies were used: anti-p62 (Enzo Life Sciences, Farmingdale, NY, USA, PM045, 1:1000), anti-LC3B (Sigma-Aldrich, L7543, 1:1000), anti-ubiquitin (Cell Signaling Technology, #2118, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, #2118, 1:2000), anti-histone H3 (Cell Signaling Technology, #9715, 1:1000), and anti-lamin A/C (Merck Millipore, Temecula, CA, USA, #05-714, 1:1000).

    Techniques: Knock-Out, Staining, Confocal Microscopy, Labeling, CCK-8 Assay

    Methylmercury (MeHg) increases p62 protein in an autophagy-independent manner. ( A ) Wild-type (WT) and Atg5 knockout (KO) mouse embryonic fibroblast cells were treated with 1 µM MeHg for 0–24 h. Whole cell lysates were subjected to immunoblots for p62, LC3-I, and LC3-II. GAPDH was used as the loading control. ( B ) Blots were quantitated and relative expression values determined.

    Journal: Scientific Reports

    Article Title: Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

    doi: 10.1038/s41598-017-17112-8

    Figure Lengend Snippet: Methylmercury (MeHg) increases p62 protein in an autophagy-independent manner. ( A ) Wild-type (WT) and Atg5 knockout (KO) mouse embryonic fibroblast cells were treated with 1 µM MeHg for 0–24 h. Whole cell lysates were subjected to immunoblots for p62, LC3-I, and LC3-II. GAPDH was used as the loading control. ( B ) Blots were quantitated and relative expression values determined.

    Article Snippet: The following primary antibodies were used: anti-p62 (Enzo Life Sciences, Farmingdale, NY, USA, PM045, 1:1000), anti-LC3B (Sigma-Aldrich, L7543, 1:1000), anti-ubiquitin (Cell Signaling Technology, #2118, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, #2118, 1:2000), anti-histone H3 (Cell Signaling Technology, #9715, 1:1000), and anti-lamin A/C (Merck Millipore, Temecula, CA, USA, #05-714, 1:1000).

    Techniques: Knock-Out, Western Blot, Expressing

    Methylmercury (MeHg) induces ubiquitinated proteins and co-localizes with p62. ( A ) Confocal immunofluorescence images of wild-type and p62 knockout (KO) mouse embryonic fibroblasts (MEFs) stained for p62 (green) and ubiquitinated (red) proteins. Scale bars: 20 µm. ( B ) Wild-type MEFs were lysed with Triton X-100 buffer and 1 mg of protein was immunoprecipitated with a p62 antibody. The interaction was determined by immunoblot analyses for p62 and ubiquitin. ( C ) Confocal immunofluorescence images of wild-type MEFs stained for p62 and ubiquitin. Cells were treated with 20 µM chloroquine (CQ) for 6 h or with 1 µM MeHg for 18 h and 20 µM CQ for 6 h. Scale bars: 20 µm.

    Journal: Scientific Reports

    Article Title: Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

    doi: 10.1038/s41598-017-17112-8

    Figure Lengend Snippet: Methylmercury (MeHg) induces ubiquitinated proteins and co-localizes with p62. ( A ) Confocal immunofluorescence images of wild-type and p62 knockout (KO) mouse embryonic fibroblasts (MEFs) stained for p62 (green) and ubiquitinated (red) proteins. Scale bars: 20 µm. ( B ) Wild-type MEFs were lysed with Triton X-100 buffer and 1 mg of protein was immunoprecipitated with a p62 antibody. The interaction was determined by immunoblot analyses for p62 and ubiquitin. ( C ) Confocal immunofluorescence images of wild-type MEFs stained for p62 and ubiquitin. Cells were treated with 20 µM chloroquine (CQ) for 6 h or with 1 µM MeHg for 18 h and 20 µM CQ for 6 h. Scale bars: 20 µm.

    Article Snippet: The following primary antibodies were used: anti-p62 (Enzo Life Sciences, Farmingdale, NY, USA, PM045, 1:1000), anti-LC3B (Sigma-Aldrich, L7543, 1:1000), anti-ubiquitin (Cell Signaling Technology, #2118, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, #2118, 1:2000), anti-histone H3 (Cell Signaling Technology, #9715, 1:1000), and anti-lamin A/C (Merck Millipore, Temecula, CA, USA, #05-714, 1:1000).

    Techniques: Immunofluorescence, Knock-Out, Staining, Immunoprecipitation

    Methylmercury (MeHg) leads to increased p62 mRNA and protein levels in mouse embryonic fibroblasts (MEFs). ( A ) mRNA levels of p62 relative to GAPDH after treatment with 1 µM MeHg for 0-24 h. Data are expressed as means ± SEM for 3 independent experiments. ** p

    Journal: Scientific Reports

    Article Title: Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

    doi: 10.1038/s41598-017-17112-8

    Figure Lengend Snippet: Methylmercury (MeHg) leads to increased p62 mRNA and protein levels in mouse embryonic fibroblasts (MEFs). ( A ) mRNA levels of p62 relative to GAPDH after treatment with 1 µM MeHg for 0-24 h. Data are expressed as means ± SEM for 3 independent experiments. ** p

    Article Snippet: The following primary antibodies were used: anti-p62 (Enzo Life Sciences, Farmingdale, NY, USA, PM045, 1:1000), anti-LC3B (Sigma-Aldrich, L7543, 1:1000), anti-ubiquitin (Cell Signaling Technology, #2118, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, #2118, 1:2000), anti-histone H3 (Cell Signaling Technology, #9715, 1:1000), and anti-lamin A/C (Merck Millipore, Temecula, CA, USA, #05-714, 1:1000).

    Techniques:

    Co-localization of LC3 with ubiquitinated proteins is impaired in the absence of p62. Wild-type and p62 knockout (KO) mouse embryonic fibroblasts (MEFs) were immunostained for endogenous LC3 (green) and ubiquitinated proteins (red), and imaged using confocal microscopy. WT and p62KO MEFs were treated with 20 µM chloroquine (CQ) for 6 h ( A , C ) or with 1 µM MeHg for 18 h before incubation with 20 µM CQ for 6 h ( B , D ). The boxed areas in the composite images are shown at a higher magnification. Scale bars: 20 µm. ( E ) Quantification of the number of colocalizations was performed in 20 cells. Results are shown as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Sequestosome1/p62 protects mouse embryonic fibroblasts against low-dose methylercury-induced cytotoxicity and is involved in clearance of ubiquitinated proteins

    doi: 10.1038/s41598-017-17112-8

    Figure Lengend Snippet: Co-localization of LC3 with ubiquitinated proteins is impaired in the absence of p62. Wild-type and p62 knockout (KO) mouse embryonic fibroblasts (MEFs) were immunostained for endogenous LC3 (green) and ubiquitinated proteins (red), and imaged using confocal microscopy. WT and p62KO MEFs were treated with 20 µM chloroquine (CQ) for 6 h ( A , C ) or with 1 µM MeHg for 18 h before incubation with 20 µM CQ for 6 h ( B , D ). The boxed areas in the composite images are shown at a higher magnification. Scale bars: 20 µm. ( E ) Quantification of the number of colocalizations was performed in 20 cells. Results are shown as mean ± SEM.

    Article Snippet: The following primary antibodies were used: anti-p62 (Enzo Life Sciences, Farmingdale, NY, USA, PM045, 1:1000), anti-LC3B (Sigma-Aldrich, L7543, 1:1000), anti-ubiquitin (Cell Signaling Technology, #2118, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, #2118, 1:2000), anti-histone H3 (Cell Signaling Technology, #9715, 1:1000), and anti-lamin A/C (Merck Millipore, Temecula, CA, USA, #05-714, 1:1000).

    Techniques: Knock-Out, Confocal Microscopy, Incubation

    Super-resolution microscopy (SR-SIM) revealed that the aggresome consists of p62 bodies containing p62 and ubiquitinated proteins. N2a cells stably expressing RFP-fused ubiquitin (R-UB) were treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA for 12 h as indicated. S403-phosphorylated p62 (S403-P; green) in ( A ), Lamp2A (lysosome; green) in ( B ), RFP-UB (red), and total p62 (blue) in ( A,B ) were visualized by super-resolution structured illumination microscopy (SR-SIM) as indicated. Magnified images in square box (10 µm each side) on left panels are shown. Scale bars represent 5 µm in wide images or 2 µm in magnified images as indicated.

    Journal: Scientific Reports

    Article Title: N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

    doi: 10.1038/s41598-018-27872-6

    Figure Lengend Snippet: Super-resolution microscopy (SR-SIM) revealed that the aggresome consists of p62 bodies containing p62 and ubiquitinated proteins. N2a cells stably expressing RFP-fused ubiquitin (R-UB) were treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA for 12 h as indicated. S403-phosphorylated p62 (S403-P; green) in ( A ), Lamp2A (lysosome; green) in ( B ), RFP-UB (red), and total p62 (blue) in ( A,B ) were visualized by super-resolution structured illumination microscopy (SR-SIM) as indicated. Magnified images in square box (10 µm each side) on left panels are shown. Scale bars represent 5 µm in wide images or 2 µm in magnified images as indicated.

    Article Snippet: Anti-p62 (polyclonal PM045 for western blotting, and polyclonal C-terminal p62 PM066 for immunofluorescence, MBL International), anti-multiubiquitin (FK2, MBL International), anti-Lamp-2 (ABL-93, Southern Biotech, Birmingham, AL, USA), anti-γ-tubulin (GTU-88, Sigma), anti-ubiquitin (monomer; polyclonal, DAKO, Glostrup, Denmark), anti-HDAC6 (D-11, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (Wako) were purchased from the indicated vendors.

    Techniques: Microscopy, Stable Transfection, Expressing

    AcylDAs induce aggresome formation without proteasome inhibition. ( A ) N2a cells stably expressing GFP-vimentin were treated with 5 µM OLDA, 5 µM NADA, 10 µM anandamide (AEA), 50 µM capsaicin, or 1 µM MG132 for 12 h as indicated. G-vimentin (G-Vim, green in merged images), p62 (red), and DAPI (blue; nucleus) were visualized by fluorescence microscopy. The arrows indicate aggresomes surrounded by the vimentin cage. ( B ) GFP-vimentin cell lines were treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA and with or without 10 µM nocodazole (+NOC) simultaneously for 24 h as indicated. G-Vimentin (G-Vim, green), p62 (red), and S403-phospho-p62 (S403-P; blue) were visualized by confocal microscopy. Scale bars represent 5 µm.

    Journal: Scientific Reports

    Article Title: N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

    doi: 10.1038/s41598-018-27872-6

    Figure Lengend Snippet: AcylDAs induce aggresome formation without proteasome inhibition. ( A ) N2a cells stably expressing GFP-vimentin were treated with 5 µM OLDA, 5 µM NADA, 10 µM anandamide (AEA), 50 µM capsaicin, or 1 µM MG132 for 12 h as indicated. G-vimentin (G-Vim, green in merged images), p62 (red), and DAPI (blue; nucleus) were visualized by fluorescence microscopy. The arrows indicate aggresomes surrounded by the vimentin cage. ( B ) GFP-vimentin cell lines were treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA and with or without 10 µM nocodazole (+NOC) simultaneously for 24 h as indicated. G-Vimentin (G-Vim, green), p62 (red), and S403-phospho-p62 (S403-P; blue) were visualized by confocal microscopy. Scale bars represent 5 µm.

    Article Snippet: Anti-p62 (polyclonal PM045 for western blotting, and polyclonal C-terminal p62 PM066 for immunofluorescence, MBL International), anti-multiubiquitin (FK2, MBL International), anti-Lamp-2 (ABL-93, Southern Biotech, Birmingham, AL, USA), anti-γ-tubulin (GTU-88, Sigma), anti-ubiquitin (monomer; polyclonal, DAKO, Glostrup, Denmark), anti-HDAC6 (D-11, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (Wako) were purchased from the indicated vendors.

    Techniques: Inhibition, Stable Transfection, Expressing, Fluorescence, Microscopy, Confocal Microscopy

    p62/SQSTM1 is essential for ubiquitinated protein accumulation in aggresomes, but is not required for vimentin cage formation. Wild-type MEF cells ( A ), p62 knockout MEF cells ( B ), or p62KO + GFP-p62 MEF cells ( C ) were treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA for 12 h as indicated. Total p62 (green), ubiquitin monomer (UB (mono), red), and DAPI (nucleus, blue) were visualized by confocal microscopy as indicated. ( D ) GFP-Vimentin plasmid was transiently transfected in wild-type MEF or p62KO MEF cells for 24 h and treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA for 12 h as indicated. GFP-Vimentin (green), multi-ubiquitin (UB (FK2), red), and p62 (blue) were visualized by confocal microscopy and merged images are shown as indicated. Scale bars represent 5 µm.

    Journal: Scientific Reports

    Article Title: N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

    doi: 10.1038/s41598-018-27872-6

    Figure Lengend Snippet: p62/SQSTM1 is essential for ubiquitinated protein accumulation in aggresomes, but is not required for vimentin cage formation. Wild-type MEF cells ( A ), p62 knockout MEF cells ( B ), or p62KO + GFP-p62 MEF cells ( C ) were treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA for 12 h as indicated. Total p62 (green), ubiquitin monomer (UB (mono), red), and DAPI (nucleus, blue) were visualized by confocal microscopy as indicated. ( D ) GFP-Vimentin plasmid was transiently transfected in wild-type MEF or p62KO MEF cells for 24 h and treated with 1 µM MG132, 5 µM OLDA, or 5 µM NADA for 12 h as indicated. GFP-Vimentin (green), multi-ubiquitin (UB (FK2), red), and p62 (blue) were visualized by confocal microscopy and merged images are shown as indicated. Scale bars represent 5 µm.

    Article Snippet: Anti-p62 (polyclonal PM045 for western blotting, and polyclonal C-terminal p62 PM066 for immunofluorescence, MBL International), anti-multiubiquitin (FK2, MBL International), anti-Lamp-2 (ABL-93, Southern Biotech, Birmingham, AL, USA), anti-γ-tubulin (GTU-88, Sigma), anti-ubiquitin (monomer; polyclonal, DAKO, Glostrup, Denmark), anti-HDAC6 (D-11, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (Wako) were purchased from the indicated vendors.

    Techniques: Knock-Out, Confocal Microscopy, Plasmid Preparation, Transfection

    AcylDA induce p62 expression in transcription level but does not affect the expression of other autophagy receptors. ( A ) Relative mRNA levels of autophagy receptors in N2a cells treated with 5 µM OLDA, 5 µM NADA, 10 µM anandamide (AEA), 50 µM capsaicin (Caps), or 1 µM MG132 for 12 h as indicated were measured by quantitative RT-PCR (qPCR) analysis. mRNA amounts of each autophagy receptor were normalized to Gapdh. ( B ) Relative mRNA level of LC3B in N2a cells treated with 5 µM OLDA or 5 µM NADA for 24 h was measured by qPCR and normalized to Gapdh. ( C ) Relative mRNA levels of Nqo1 in N2a cells treated with 5 µM OLDA or 5 µM NADA for 12 h were measured by quantitative RT-PCR and the amount was normalized to Gapdh. Error bars represent SEM (n = 4), and p-value (Student’s t -test) is shown. *and ***represent p

    Journal: Scientific Reports

    Article Title: N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

    doi: 10.1038/s41598-018-27872-6

    Figure Lengend Snippet: AcylDA induce p62 expression in transcription level but does not affect the expression of other autophagy receptors. ( A ) Relative mRNA levels of autophagy receptors in N2a cells treated with 5 µM OLDA, 5 µM NADA, 10 µM anandamide (AEA), 50 µM capsaicin (Caps), or 1 µM MG132 for 12 h as indicated were measured by quantitative RT-PCR (qPCR) analysis. mRNA amounts of each autophagy receptor were normalized to Gapdh. ( B ) Relative mRNA level of LC3B in N2a cells treated with 5 µM OLDA or 5 µM NADA for 24 h was measured by qPCR and normalized to Gapdh. ( C ) Relative mRNA levels of Nqo1 in N2a cells treated with 5 µM OLDA or 5 µM NADA for 12 h were measured by quantitative RT-PCR and the amount was normalized to Gapdh. Error bars represent SEM (n = 4), and p-value (Student’s t -test) is shown. *and ***represent p

    Article Snippet: Anti-p62 (polyclonal PM045 for western blotting, and polyclonal C-terminal p62 PM066 for immunofluorescence, MBL International), anti-multiubiquitin (FK2, MBL International), anti-Lamp-2 (ABL-93, Southern Biotech, Birmingham, AL, USA), anti-γ-tubulin (GTU-88, Sigma), anti-ubiquitin (monomer; polyclonal, DAKO, Glostrup, Denmark), anti-HDAC6 (D-11, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (Wako) were purchased from the indicated vendors.

    Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Drag screening for p62 promoter activation compounds. ( A ) A scheme of p62 promoter (−1781 to 5′ untranslated region of p62 exon 1)-driven EGFP gene (p62PR-EGFP) and drug screening using LOPAC1280® library. The compounds that induced GFP expression and RFPu accumulation were selected and the chemical structures of OLDA, NADA, and AEA are shown ( B ). Normal N2a cells were treated with 5 µM OLDA, 5 µM NADA, 10 µM AEA, 50 µM capsaicin, or 1 µM MG132 for 12 h as indicated ( C ). N2a cells were treated with 5 µM OLDA or 5 µM NADA and with or without 1 µM bafilomycinA1 (BafA) simultaneously for 12 h. S403-phosphorylated p62 (S403-P) was detected with monoclonal anti-phospho-p62 (S403) antibody (4F6). Total p62 was visualized with anti-p62 (polyclonal). The ratio between relative amounts of phosphorylated or total p62 normalized to γ-tubulin is shown ( B,C ). ( D ) In vitro proteasome activity assay was carried out using purified yeast 26S proteasomes with 2 nM proteasome, 125 µM Suc-LLVY-AMC, and compounds as indicated incubated at 30 °C for 30 min followed by calculation of the relative proteasome activity. Data are shown as the mean ± SEM values of 9–12 measurements under each condition and p values (Student’s t -test) are shown.

    Journal: Scientific Reports

    Article Title: N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

    doi: 10.1038/s41598-018-27872-6

    Figure Lengend Snippet: Drag screening for p62 promoter activation compounds. ( A ) A scheme of p62 promoter (−1781 to 5′ untranslated region of p62 exon 1)-driven EGFP gene (p62PR-EGFP) and drug screening using LOPAC1280® library. The compounds that induced GFP expression and RFPu accumulation were selected and the chemical structures of OLDA, NADA, and AEA are shown ( B ). Normal N2a cells were treated with 5 µM OLDA, 5 µM NADA, 10 µM AEA, 50 µM capsaicin, or 1 µM MG132 for 12 h as indicated ( C ). N2a cells were treated with 5 µM OLDA or 5 µM NADA and with or without 1 µM bafilomycinA1 (BafA) simultaneously for 12 h. S403-phosphorylated p62 (S403-P) was detected with monoclonal anti-phospho-p62 (S403) antibody (4F6). Total p62 was visualized with anti-p62 (polyclonal). The ratio between relative amounts of phosphorylated or total p62 normalized to γ-tubulin is shown ( B,C ). ( D ) In vitro proteasome activity assay was carried out using purified yeast 26S proteasomes with 2 nM proteasome, 125 µM Suc-LLVY-AMC, and compounds as indicated incubated at 30 °C for 30 min followed by calculation of the relative proteasome activity. Data are shown as the mean ± SEM values of 9–12 measurements under each condition and p values (Student’s t -test) are shown.

    Article Snippet: Anti-p62 (polyclonal PM045 for western blotting, and polyclonal C-terminal p62 PM066 for immunofluorescence, MBL International), anti-multiubiquitin (FK2, MBL International), anti-Lamp-2 (ABL-93, Southern Biotech, Birmingham, AL, USA), anti-γ-tubulin (GTU-88, Sigma), anti-ubiquitin (monomer; polyclonal, DAKO, Glostrup, Denmark), anti-HDAC6 (D-11, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (Wako) were purchased from the indicated vendors.

    Techniques: Activation Assay, Expressing, In Vitro, Activity Assay, Purification, Incubation

    AcylDA accelerates huntingtin aggregation. ( A ) N2a cells stably expressing huntingtin-ex1-Q148-YFP (N2a HttQ148-YFP) were treated with 3 µM OLDA for 3 days after Htt-YFP induction by doxycycline. Cells with Htt aggregates were counted (n > 100) and the number of aggregates was divided by the number of nuclei. ( B ) N2a HttQ148-YFP cells were treated with 5 µM OLDA, 5 µM NADA, 10 µM AEA, 50 µM capsaicin, or 1 µM MG132 for 48 h as indicated. Htt aggregates were visualized with anti-GFP antibody by filter trap assay. As a sample control, dot blot of the same sample on the polyvinylidene fluoride membrane is shown. The relative amount of HttQ148-YFP is shown. Error bars represent SEM (n = 4) and p values (Student’s t- test) are shown. ( C ) N2a cells were treated with 0.2 µM MG132 and/or 5 µM OLDA or 5 µM NADA for 12 h as indicated. Total ubiquitinated proteins (polyUb; monomer), S403-phospho-p62 (S403-P), total p62, and β-actin are shown as indicated. Relative amounts of polyubiquitinated proteins (polyUb) in duplicated analysis are depicted. Error bars represent SD (n = 2).

    Journal: Scientific Reports

    Article Title: N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

    doi: 10.1038/s41598-018-27872-6

    Figure Lengend Snippet: AcylDA accelerates huntingtin aggregation. ( A ) N2a cells stably expressing huntingtin-ex1-Q148-YFP (N2a HttQ148-YFP) were treated with 3 µM OLDA for 3 days after Htt-YFP induction by doxycycline. Cells with Htt aggregates were counted (n > 100) and the number of aggregates was divided by the number of nuclei. ( B ) N2a HttQ148-YFP cells were treated with 5 µM OLDA, 5 µM NADA, 10 µM AEA, 50 µM capsaicin, or 1 µM MG132 for 48 h as indicated. Htt aggregates were visualized with anti-GFP antibody by filter trap assay. As a sample control, dot blot of the same sample on the polyvinylidene fluoride membrane is shown. The relative amount of HttQ148-YFP is shown. Error bars represent SEM (n = 4) and p values (Student’s t- test) are shown. ( C ) N2a cells were treated with 0.2 µM MG132 and/or 5 µM OLDA or 5 µM NADA for 12 h as indicated. Total ubiquitinated proteins (polyUb; monomer), S403-phospho-p62 (S403-P), total p62, and β-actin are shown as indicated. Relative amounts of polyubiquitinated proteins (polyUb) in duplicated analysis are depicted. Error bars represent SD (n = 2).

    Article Snippet: Anti-p62 (polyclonal PM045 for western blotting, and polyclonal C-terminal p62 PM066 for immunofluorescence, MBL International), anti-multiubiquitin (FK2, MBL International), anti-Lamp-2 (ABL-93, Southern Biotech, Birmingham, AL, USA), anti-γ-tubulin (GTU-88, Sigma), anti-ubiquitin (monomer; polyclonal, DAKO, Glostrup, Denmark), anti-HDAC6 (D-11, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (Wako) were purchased from the indicated vendors.

    Techniques: Stable Transfection, Expressing, TRAP Assay, Dot Blot