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  • 99
    Millipore anti p62 sqstm1 antibody
    PPARα or FXR agonist affects autophagic flux in murine hepatocytes a , Autophagic flux was assessed by LC3-Immunoblot analysis in AML12 cells treated with indicated dose of Wy or co-treated with Wy and bafilomycin A1 (BafA1). b and e , Biochemical determination of autophagy <t>(p62/SQSTM1</t> immunoblot) in AML12 cells treated with indicated doses of Wy-14,643 (Wy) or GW4064 for 24 h. GW4064-treated cells were starved for 2 h. c , Primary hepatocytes prepared from GFP-LC3 Tg/+ mice were treated with indicated doses of Torin1 or GW7647. GFP-LC3 cleavage was assessed by GFP-Immunoblot analysis. d , LC3-Immunoblot in AML12 cells treated with indicated doses of GW4064 or co-treated with GW4064 and Torin1. All drug treatments were done in complete media for 24 h otherwise indicated in each panel. β-actin is a loading control. f , Quantification of autophagic flux shown in Fig. 1a . Numbers of autolysosomes (ALs) induced by 2 h starvation + Veh were set as 100%. Numbers of autolysosomes = RFP positive vesicles – GFP positive vesicles. 30 cells were counted per condition (** P
    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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    93
    Cell Signaling Technology Inc p62
    HDAC3 inhibition enhanced autophagy in diabetic mice subjected to cerebral I/R injury. Representative immunofluorescence staining images and assessment of LC3B (a, d) and beclin-1 (b, e) (×100). Representative electron micrographs of each group (c) (×5000). Representative Western blot images and assessment of <t>p62</t> (f) level in each group. All results are presented as mean ± SD, n = 6/group. # P
    P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 3597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p62  (Abcam)
    93
    Abcam p62
    Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors <t>p62,</t> BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P
    P62, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 1709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc anti p62
    Targeted cytoplasmic irradiation induces autophagy in SAE cells A. LC3B puncta formation increases in SAE cells after cytoplasmic irradiation. SAE cells were treated with 5 α-particles 8 μm away from the cell nucleus along the major axis. Cells were incubated for 4 hr (left) or indicated time (right) before fixation and immunofluorescence staining for LC3B (green). Nuclei were visualized by DAPI (blue). LC3B positive ratio was quantified by counting cells with LC3B puncta based on total number of cells per dish in each time point (n=3). Scale bar = 10 μm. B. Autophagic flux assay proves the increase of autophagosomal synthesis. SAE cells were pre-treated with DMSO as control or 100 nM Bafilomycin A1 (Baf) for 1 hr before cytoplasmic irradiation. LC3B positive ratio were determined as described in Figure 1A. Scale bar = 10 μm. C. <t>p62</t> expression reduces after cytoplasmic irradiation. SAE cells were cytoplasmic irradiated and incubated for 20 hr before fixation and immunofluorescence staining for p62 (green). Nuclei were visualized by DAPI (blue). Protein expression level was calculated using total cell fluorescence as described in Materials and Methods. Scale bar = 10 μm. D. Only cytoplasmic irradiation induces autophagy. SAE cells were irradiated with 5 α-particles through nucleus or cytoplasm. Cells were harvested 4 hr after irradiation and LC3B positive ratio were evaluated (n=3). E. Free radical scavenger attenuates autophagy induced by cytoplasmic irradiation. SAE cells were pre-treated with 0.5% DMSO or N-acetyl cysteine (NAC) for 30 min or 24 hr, respectively. LC3B positive ratio were evaluated 4 hr after cytoplasmic irradiation. CI, cytoplasmic irradiation. CTCF, corrected total cell fluorescence. Error bar ± S.D. * p
    Anti P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 831 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti p62
    Tigecycline induces autophagy of CML cells by downregulating the PI3K-ATK-mTOR signaling pathway. ( a ) Autophagic vacuoles were measured by transmission electron microscopy. Upper panel: autophagic vacuoles in CML cells with and without tigecycline treatment. Lower panel: amplified image of autophagic vacuoles in tigecycline-treated CML cells. ( b ) Confocal microscopy analysis of autophagy. Blue spots indicate nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Green spots indicate autophagic vacuoles stained with LC3B dye. ( c ) Western blot analysis to evaluate the levels of autophagy related protein <t>P62</t> and LC3B, and mTOR and its upstream regulator AKT, and downstream sensors P70S6 and 4E-BP1
    Anti P62, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc sqstm1 p62
    Autophagy promotion is involved in the anti‐AML effect of matrine in vivo . ( A ) Experimental protocol was used to assess the anti‐AML effect of matrine on C57BL/6 mice. ( B ) After treated with matrine, chloroquine (CQ) or saline (Veh), IHC staining of <t>SQSTM1/P62</t> and LC3 II in spleen and bone were observed by microscopy and representative images were showed. The scale bars are 50 μm. ( C ) IOD of SQSTM1/P62 and LC3 II of spleen and bone were showed in bar charts. ( D ) Wright's staining of peripheral blood and bone marrow was performed to observe immature leucocyte cells by microscopy, and representative images are showed. The scale bars are 10 μm, and arrows indicate immature leucocyte cells. ( E ) The percentage of immature leucocyte cells in peripheral blood and bone marrow was shown. ( F ) The spleen weight of mice was evaluated after killed. ( G ) The levels of SQSTM1/p62, LC3 II and cleaved caspase‐3 in bone marrow mononuclear cells were determined using Western blot analysis. ( H ) Kaplan–Meier curves for survival were assessed for 10 mice per group, and the long‐rank test was used to evaluate the statistical differences in survival. Results were expressed as mean ± S.E.M., and images shown were representatives of at least three independent experiments. * P
    Sqstm1 P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology p62
    H 2 S pretreatment suppressed TBI-induced the increase of Vps34 and <t>P62.</t> Sample immunoblots probed for Vps34, P62 and GAPDH are showed above. The bar chart below demonstrates the ratio of Vps34 and P62 relative to GAPDH. TBI-induced up-regulation of Vps34 and down-regulation of P62 were inhibited by H 2 S in the cortex (A) and hippocampus (B). Optical densities of the protein bands were quantitatively analyzed with Sigma Scan Pro 5 and normalized with loading control GAPDH. Semiquantitative analysis (relative optical density) of the intensity of staining of Vps34 to GAPDH in the cortex (C) and hippocampus (D). Semiquantitative analysis (relative optical density) of the intensity of staining of P62 to GAPDH in the cortex (E) and hippocampus (F). The data are means ± SEM (n = 3, *P
    P62, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam anti sqstm1 p62 antibody
    TFEB silencing counteracts trehalose-induced expression of autophagic genes. ( a-n ) NSC34 cells were transfected with Tfeb or non-targeting (as control) siRNAs and treated with 100 mM glucose or trehalose. ( a ) WB analysis was performed, and the bar graphs (b-c) represent the mean relative optical density of <t>SQSTM1/p62</t> and MAP1LC3B-II:MAP1LC3B-I protein expression levels, respectively, performed with n = 3 independent samples. LC3-II:LC3-I ratio was calculated by densitometric analysis of both bands. (*** pÂÂ
    Anti Sqstm1 P62 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson p62
    G-TPP activity is conserved in primary fibroblasts ( A , C ) Fibroblasts were treated with 15 µM G-TPP for the indicated time points. Cells were harvested and western blots were probed with antibodies against (A) PINK1, pS65-Ub and total Ub or (C) autophagy adapter proteins. GAPDH and Vinculin served as loading control. G-TPP treatment led to PINK1 stabilization and pS65-Ub induction in primary skin fibroblasts. <t>p62</t> levels were induced upon G-TPP treatment, while other adapters seemed decreased. ( B , D ) Human fibroblasts were treated with 15 µM G-TPP for 16 h and fixed and stained with antibodies against (B) pS65-Ub (green) or (D) the autophagy adapters NBR1, NDP52, p62, OPTN and TAX1BP1 (green). Mitochondria were stained with antibodies against TOM20 (red), nuclei were visualized with Hoechst (blue). Scale bars indicate 10 µM. A magnified image of the boxed region, the fluorescence profile along the arrow and the Pearson’s correlation coefficient of adapter protein and mitochondrial stainingare shown to the right. Shown is the mean ± SEM of at least five randomly selected images (unpaired, two-sided t -test, *** p
    P62, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti p62
    The antiviral effect of ANKS4B was exerted through inhibiting the autophagy. (A) Co-IP assay. A549 and 293T cells were co-transfected with plasmids expressing ANKS4B-FLAG protein and GRP78. Whole cell extracts were prepared for Co-IP assay using anti-FLAG. (B) Control, ANKS4B-KO1, ANKS4B-KO2, and ANKS4B-RES (R) Huh7 cells were infected with ZIKV (MOI 3). Cell lysates were prepared at 24 h p.i for western blot to detect the expression levels of ZIKV E protein, LC3B, and <t>p62.</t> (C–F) Autophagy inhibitor assay. Control, ANKS4B-KO1, ANKS4B-KO2, and ANKS4B-RES cells were treated with DMSO or chloroquine (CQ, 50 μM) or 3-methyladenine (3-MA, 5 mM) after ZIKV inoculation. At 24 h p.i., cells and supernatants were harvested for western blot (C,E) or plaque assay (D,F) . Levels of p62, ZIKV E, and GAPDH were measured. All the data were shown as means ± S.D. (error bars) from at least three independent experiments. NS, not significant. * 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
    Progen Biotechnik p62
    Microglia-engulfed α-synuclein is sequestered by autophagosomes and degraded by autophagy. (a, b, c) Cultured primary microglia from WT mice (a and left panels of b) and GFP-LC3-transgenic mice (right panels of b) were treated 250nM h α-Syn protein for indicated time and stained with antibodies against human α-synuclein (MJFR1 clone, a and b), ubiquitin ( a ), or <t>p62</t> ( b ). The number of h α-Syn/ubiquitin-positive puncta was quantified ( c ). In (a), empty arrowheads indicate a portion of α-synuclein colocalized with ubiquitin-positive puncta and disappear in 24 hours. Arrows represent a portion of α-synuclein not colocalized with ubiquitin. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. Scale bar, 10 µm; 2 µm in magnified boxes. (d) At 6-weeks post-AAV- h α-Syn inoculation into GFP-LC3 mice, brain slices were fixed and stained with antibodies against human α-synuclein, GFP, and Iba-1 (left panel). 3D surface-rendering images of each protein in microglia were made with Imaris software (Bitplane, right panel). Scale bar, 10µm; 1µm in the magnified box. (e) Ultrastructure of puncta from 250nM h α-Syn protein-treated cells for 6 hours was observed by an electron microscope. Arrows indicate double-membrane structures. Scale bar, 500 nm in the left panel; 100 nm in the right panel. (f, g) Microglia were treated with 250nM h α-Syn protein for 6 hours (f) or 3 hours (g) first and added SAR405 ( f ) or Torin ( g ) was added. The number of h α-Syn/ubiquitin-positive puncta was quantified in the lower panel. p values were calculated by One-way ANOVA with Newman– Keuls post hoc test (f) and Unpaired two-tailed Student’s t-test (g). (h, i) Microglia cultured either from Atg7 flox/flox mice (left panel) from Atg14 flox/flox mice (right panel) with or without Cx3cr1 Cre expression were assayed for W.B using antibodies against ATG7 or ATG14, p62, and LC3 I/II ( h ). Cells were treated with 250nM h α-Syn protein for 24 hours and the number of h α-Syn/ubiquitin-positive puncta was quantified ( i ). p values were calculated by Mann-Whitney U test. Data are representative of at least three independent experiments.
    P62, supplied by Progen Biotechnik, used in various techniques. Bioz Stars score: 92/100, based on 545 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc p62 sqstm1
    ROS produced by up-regulated MAOA plays an essential role for androgen deprivation-induced autophagy activation, NED and anti-apoptosis. ( A ) Immunoblotting of MAOA, LC3B-II and <t>p62/SQSTM1</t> in shCtrl and shMAOA LNCaP cells treated as described in Fig. 3C for 48 and 96 hours. β-actin was used as loading control. Uncropped images are presented in Supplementary Figure S13A (right). ( B ) ROS production measured by OxiSelect In Vitro ROS/RNS Assay Kit in cells treated as described in ( A ). Data represent mean ± S.D. (n = 3); *** p
    P62 Sqstm1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 314 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam sqstm1 p62
    Morphine induces the degradation of PINK1 and the rescue of PINK1 improves the accumulation of <t>SQSTM1/p62</t> induced by morphine. ( A ) SH-SY5Y cells were treated with CCCP (10 μM) for 2.5 h with or without morphine (200 μM) for 24 h. The level of PINK1 was analyzed by western blotting. n = 3, *** P
    Sqstm1 P62, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology p62 sqstm1
    Model for HSF1 and autophagy in cellular response to Hsp90 inhibitors. HSF1 is activated by Hsp90 inhibitors and enhances the activity of the transcription factor HSF1, which by promoting the expression of <t>p62/SQSTM1</t> maintains autophagic and mitigates
    P62 Sqstm1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti p62
    Momordicoside G selectively affects M1 macrophage phenotype and function in vitro . (A) Macrophage phenotypes analyzed by FITC-conjugated anti-mouse iNOS or arginase staining ( n = 10, 40×). (B) Cell viability examined by MTT ( n = 5). (C) ROS detected by DCFH-DA ( n = 5). (D) Cell autophagy-associated markers examined by Western blot ( n = 3). (E) Cell autophagy indicated by AO, LC3-B, and <t>P62</t> staining ( n = 5, 40×). The data presents mean ± SD, the experiments were repeated 3 times, and statistical significance was determined by a t -test. ∗ P
    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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    p62  (Abnova)
    93
    Abnova p62
    <t>p62</t> and LC3 in freshly isolated human endothelial cells. A : Venous endothelial cells were isolated from healthy subjects and diabetic subjects as outlined in the methods. Endothelial cells from diabetic subjects (n=11) and non-diabetic controls (n=16)
    P62, supplied by Abnova, used in various techniques. Bioz Stars score: 93/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Proteintech rabbit p62 sqstm1 polyclonal
    <t>p62</t> and LC3 in freshly isolated human endothelial cells. A : Venous endothelial cells were isolated from healthy subjects and diabetic subjects as outlined in the methods. Endothelial cells from diabetic subjects (n=11) and non-diabetic controls (n=16)
    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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    Millipore p62
    Exercise, AICAR and rapamycin counteract muscle wasting in cancer cachexia and modulate an adequate autophagic flux level. ( A ) In physiological conditions, a balance between autophagosome production and clearance maintains an adequate autophagic flux (red arrow) in the muscles, mirrored by basal levels of LC3bII (black dots) and <t>p62</t> (green dots) expression. In cachectic muscles from C26-bearing mice ( B ), a strong accumulation of both LC3bII and p62 proteins points to an unbalanced autophagosome production/clearance ratio. This correlates with the pathophysiological features observed in cancer-related muscle wasting, including overexpression of Atrogin1 and Murf1 genes, as well as a decline in body weight (BD), muscle weight (MW), fiber size and muscle function. Spontaneous wheel running, or treatment with AICAR or rapamycin ( C ) counteracts cancer-related muscle wasting in tumor-bearing mice and induces a decrease in both LC3bII and p62 accumulation. Atrogin1 and Murf1 gene expression was restored to the basal levels observed in healthy muscles. This is associated with increased body and muscle weight and improved muscle function.
    P62, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 743 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem p62
    Increased <t>p62</t> expression of HCC and CCA patients carrying the rs146589465-G variant. Sections from 28 paraffin-embedded HCC or CCA patient samples were stained with a p62 antibody. Numbers correlate with patient numbering in Table S20 and an asterisk indicates an rs146589465-G carrier. Information on liver disease is indicated; Cirr, cirrhosis; H.C., hemochromatosis; HBV, Hepatitis B virus. Samples are grouped by International Classification of Diseases (ICD) diagnoses information from the Icelandic Cancer Registry.
    P62, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology sqstm1 p62
    The MG-132-mediated upregulation of <t>SQSTM1/p62</t> protein expression requires the specific presence of ELAVL1/HuR protein. Representative western blotting (upper) and densitometric analysis (lower) of ELAVL1/HuR (A) and SQSTM1/p62 (B) proteins in the total homogenates of ELAVL1/HuR silenced ARPE-19 cells and negative control (NEG-siRNA) cells after exposure to 5 µM MG-132 for 24 h. α-tubulin was used as a loading control. Results are expressed as means ± S.E.M. *p
    Sqstm1 P62, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti p62
    Impaired autophagosome maturation leads to locomotion defects in 2-d-old adult flies. (A and B) Atg8a-positive autophagosomes and <t>p62</t> aggregates accumulate in Syx17 mutant brains (B) compared with similarly aged controls (A). (C–E) No autophagosomes are found by EM in neurons of control adult flies (C). Large-scale accumulation of autophagosomes (arrowheads) is obvious in Syx17 mutant neurons (D). Autophagosome accumulation is rescued in Syx17 mutant neurons by transgenic expression of Syx17 (E). (F and G) Quantification of data presented in A and B (F) and C–E (G); n = 9 for A and B, and n = 4 for C–E. Error bars mark SDs. ***, P
    Rabbit Anti P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Progen Biotechnik anti p62
    Autophagy is suppressed in liver macrophages after chronic ethanol exposure. Liver macrophages were isolated from WT mice after 6 weeks of a paired liquid diet or a Lieber–DeCarli diet containing 5% ethanol (v/v). A , the experimental design for B and C. B , LC3B protein expression was determined by Western blotting. LC3BII levels were reduced in liver macrophages from ethanol-fed mice. C , isolated liver macrophages were treated with ammonium chloride and leupeptin ( AC/Leu ) for 2 h, and LC3B and <t>p62</t> expression was examined by Western blotting. Similar results were obtained in four independent experiments. Representative results and their quantification are shown. *, p
    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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    Abcam mouse anti p62
    Immunohistochemical staining and double-immunofluorescence labeled microscopic images of ECIs in RCCs. Immunohistochemical staining showed that the ECIs were immunopositive for <t>P62</t> ( A ), NBR1 ( B ), LC3 ( C ), BECN1 ( D ), and ATG5 ( E ), and immunonegative for LCK (arrows in G ). Ub1 immunopositivity was detected in a small number of ECIs (arrows in F ). Double-immunofluorescence staining of ECIs demonstrated co-localization of P62 with NBR1, LC3, BECN1 or ATG5 in ECIs ( H ).
    Mouse Anti P62, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 186 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 186 article reviews
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    89
    Progen Biotechnik guinea pig anti p62
    ML-9 blocks autophagic flux. ( a ) Chloroquine does not increase LC3-II levels induced by elevated concentrations of ML-9. LNCaP cells were incubated in full, serum-starved or ML-9-containing media for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Densitometric quantitation showing autophagic flux (change in LC-II levels induced by chloroquine) is represented. Values represent means±S.E.M. n =3. ( b ) Chloroquine fails to increase the number of eGFP-positive puncta following ML-9 treatment. eGFP-LC3 expressing LNCaP cells were treated with full, serum-starved or 30 μ M ML-9-containing media for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Quantitation shown on the right represents means±S.D. GFP-positive puncta per cell ( n =50) from three independent experiments. ( c ) ML-9 induces <t>p62</t> accumulation. LNCaP cells were treated with full, serum-starved or ML-9 containing media for 12 h. Densitometric quantitation for normalized p62 relative to Actin is shown. Values represent means±S.E.M. n =3. ( d ) ML-9 blocks starvation induced autophagic flux. LNCaP cells were treated with full, serum-starved, 30 μ M ML-9-containing full medium or 30 μ M ML-9-containing serum-starved medium for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Densitometric quantitation showing autophagic flux (change in LC-II levels induced by chloroquine) as well as normalized p62 relative to Actin is shown. Values represent means±S.E.M. n =3
    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
    https://www.bioz.com/result/guinea pig anti p62/product/Progen Biotechnik
    Average 89 stars, based on 179 article reviews
    Price from $9.99 to $1999.99
    guinea pig anti p62 - by Bioz Stars, 2020-09
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    Image Search Results


    PPARα or FXR agonist affects autophagic flux in murine hepatocytes a , Autophagic flux was assessed by LC3-Immunoblot analysis in AML12 cells treated with indicated dose of Wy or co-treated with Wy and bafilomycin A1 (BafA1). b and e , Biochemical determination of autophagy (p62/SQSTM1 immunoblot) in AML12 cells treated with indicated doses of Wy-14,643 (Wy) or GW4064 for 24 h. GW4064-treated cells were starved for 2 h. c , Primary hepatocytes prepared from GFP-LC3 Tg/+ mice were treated with indicated doses of Torin1 or GW7647. GFP-LC3 cleavage was assessed by GFP-Immunoblot analysis. d , LC3-Immunoblot in AML12 cells treated with indicated doses of GW4064 or co-treated with GW4064 and Torin1. All drug treatments were done in complete media for 24 h otherwise indicated in each panel. β-actin is a loading control. f , Quantification of autophagic flux shown in Fig. 1a . Numbers of autolysosomes (ALs) induced by 2 h starvation + Veh were set as 100%. Numbers of autolysosomes = RFP positive vesicles – GFP positive vesicles. 30 cells were counted per condition (** P

    Journal: Nature

    Article Title: Nutrient Sensing Nuclear Receptors Coordinate Autophagy

    doi: 10.1038/nature13961

    Figure Lengend Snippet: PPARα or FXR agonist affects autophagic flux in murine hepatocytes a , Autophagic flux was assessed by LC3-Immunoblot analysis in AML12 cells treated with indicated dose of Wy or co-treated with Wy and bafilomycin A1 (BafA1). b and e , Biochemical determination of autophagy (p62/SQSTM1 immunoblot) in AML12 cells treated with indicated doses of Wy-14,643 (Wy) or GW4064 for 24 h. GW4064-treated cells were starved for 2 h. c , Primary hepatocytes prepared from GFP-LC3 Tg/+ mice were treated with indicated doses of Torin1 or GW7647. GFP-LC3 cleavage was assessed by GFP-Immunoblot analysis. d , LC3-Immunoblot in AML12 cells treated with indicated doses of GW4064 or co-treated with GW4064 and Torin1. All drug treatments were done in complete media for 24 h otherwise indicated in each panel. β-actin is a loading control. f , Quantification of autophagic flux shown in Fig. 1a . Numbers of autolysosomes (ALs) induced by 2 h starvation + Veh were set as 100%. Numbers of autolysosomes = RFP positive vesicles – GFP positive vesicles. 30 cells were counted per condition (** P

    Article Snippet: Materials Reagents were obtained from the following sources: C57BL/6J mice and PPARα−/− mice from the Jackson Laboratory; FXR−/− and GFP-LC3 transgenic (Tg) mice were previously described , ; Antibodies to LC3 (NB600–1384) from Novus Biologicals; rabbit antibodies to phospho-Ser240/244 S6 (cat. # 5364), S6 (cat. # 2217), phospho-Thr37/46–4E–BP1 (cat. # 2855), 4E–BP1 (cat. # 9452), anti-mouse IgG-HRP (cat. # 7076) and β-actin (cat. # 5125) from Cell Signaling Technology; antibody to normal rabbit IgG (sc-2027), PPARα (sc-9000x), FXR (sc-13063x), Pol II (sc-899x), p300 (sc-585x), CRTC2/TORC2 (sc-6714x) and anti-rabbit IgG-HRP (sc-2370) from Santa Cruz Biotechnology; antibody to Histone H3K27me3 (cat. # 39155) from ACTIVE MOTIF; GW7647 and Wy-14,643 from Cayman chemicals; GW4064 from Tocris bioscience; Bafilomycin A1 from Enzo Life Science; AML12 cells from ATCC (CRL-2254); GFP antibody (cat. # 11814460001), PhosSTOP (Cat. # 04906837001), and Complete Protease Cocktail (Cat. # 11836170001) from Roche; ITS (Cat. # 41400–045), Lipofectamine 2000 (Cat. # 11668019), BODIPY 493/503 (D-3922), Alexa Fluor 594 goat anti-rabbit IgG (Cat. # A11037), Stealth siRNAs-NCoR (NcoR1, MSS208758), SMRT (NcoR2, MSS20912), SHP (NR0b2, MSS239500), and HIGH_GC from Invitrogen; mRFP-GFP-LC3(ptfLC3) plasmid, a gift of T. Yoshimori (Addgene plasmid # 21074); anti-rabbit p62/SQSTM1 (Cat. # P0067), anti-rabbit LC3B (Cat. # L7543), Oleic-Acid-Albumin (Cat. # O3008–5ML), and Dexamethasone (Cat. # D4902–25MG) from Sigma; Protein A Sepharose CL-48 (Cat. # 17–0780–01) from GE Healthcare; VECTASHIELD (Cat. # H-1200) from Vector Laboratories; SacI-HF (Cat. # R3156S) and BglII (Cat. # R0144S) from New England Biolabs; Pierce™ BCA Protein Assay Kit (Cat. # 23227) from Thermo Scientific; SMRT antibody (ab24551) and β-hydroxybutyrate assay kit (ab83390).from abcam; anti-acetyl-Histone H4 antibody (06–866) from EMD Millipore.

    Techniques: Mouse Assay

    HDAC3 inhibition enhanced autophagy in diabetic mice subjected to cerebral I/R injury. Representative immunofluorescence staining images and assessment of LC3B (a, d) and beclin-1 (b, e) (×100). Representative electron micrographs of each group (c) (×5000). Representative Western blot images and assessment of p62 (f) level in each group. All results are presented as mean ± SD, n = 6/group. # P

    Journal: Journal of Diabetes Research

    Article Title: Inhibition of HDAC3 Ameliorates Cerebral Ischemia Reperfusion Injury in Diabetic Mice In Vivo and In Vitro

    doi: 10.1155/2019/8520856

    Figure Lengend Snippet: HDAC3 inhibition enhanced autophagy in diabetic mice subjected to cerebral I/R injury. Representative immunofluorescence staining images and assessment of LC3B (a, d) and beclin-1 (b, e) (×100). Representative electron micrographs of each group (c) (×5000). Representative Western blot images and assessment of p62 (f) level in each group. All results are presented as mean ± SD, n = 6/group. # P

    Article Snippet: The membrane was blocked with 5% BSA for 1 h and incubated with the following primary rabbit monoclonal antibodies: HDAC3, Bmal1, and p62 (1 : 1000, Cell Signaling Technology, USA) diluted in 5% w /v BSA overnight at 4°C.

    Techniques: Inhibition, Mouse Assay, Immunofluorescence, Staining, Western Blot

    Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P

    Article Snippet: The protein was boiled and subjected to western blot with antibodies against CFTR, DNMT1, DNMT3a, DNMT3b, EZH2, p62, BECN1, microtubule-associated protein 1 light chain 3 (LC3), Atg12 and β-actin (all from Abcam Inc., Cambridge, MA, USA) respectively.

    Techniques: Knock-Out, Mouse Assay, Small Interfering RNA, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Infection

    Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P

    Article Snippet: The protein was boiled and subjected to western blot with antibodies against CFTR, DNMT1, DNMT3a, DNMT3b, EZH2, p62, BECN1, microtubule-associated protein 1 light chain 3 (LC3), Atg12 and β-actin (all from Abcam Inc., Cambridge, MA, USA) respectively.

    Techniques: Mouse Assay, Knock-Out, Concentration Assay, AST Assay, Staining, Transmission Assay, Microscopy, Transmission Electron Microscopy, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay, Enzyme-linked Immunosorbent Assay, Transfection

    Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P

    Article Snippet: The protein was boiled and subjected to western blot with antibodies against CFTR, DNMT1, DNMT3a, DNMT3b, EZH2, p62, BECN1, microtubule-associated protein 1 light chain 3 (LC3), Atg12 and β-actin (all from Abcam Inc., Cambridge, MA, USA) respectively.

    Techniques: Knock-Out, Mouse Assay, Quantitative RT-PCR, Western Blot, Expressing, Activation Assay, Inhibition, Plasmid Preparation, Sequencing, Infection, Over Expression

    Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P

    Article Snippet: The protein was boiled and subjected to western blot with antibodies against CFTR, DNMT1, DNMT3a, DNMT3b, EZH2, p62, BECN1, microtubule-associated protein 1 light chain 3 (LC3), Atg12 and β-actin (all from Abcam Inc., Cambridge, MA, USA) respectively.

    Techniques: Expressing, DNA Methylation Assay, Knock-Out, Mouse Assay, Small Interfering RNA, Methylation, Methylation Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection

    Targeted cytoplasmic irradiation induces autophagy in SAE cells A. LC3B puncta formation increases in SAE cells after cytoplasmic irradiation. SAE cells were treated with 5 α-particles 8 μm away from the cell nucleus along the major axis. Cells were incubated for 4 hr (left) or indicated time (right) before fixation and immunofluorescence staining for LC3B (green). Nuclei were visualized by DAPI (blue). LC3B positive ratio was quantified by counting cells with LC3B puncta based on total number of cells per dish in each time point (n=3). Scale bar = 10 μm. B. Autophagic flux assay proves the increase of autophagosomal synthesis. SAE cells were pre-treated with DMSO as control or 100 nM Bafilomycin A1 (Baf) for 1 hr before cytoplasmic irradiation. LC3B positive ratio were determined as described in Figure 1A. Scale bar = 10 μm. C. p62 expression reduces after cytoplasmic irradiation. SAE cells were cytoplasmic irradiated and incubated for 20 hr before fixation and immunofluorescence staining for p62 (green). Nuclei were visualized by DAPI (blue). Protein expression level was calculated using total cell fluorescence as described in Materials and Methods. Scale bar = 10 μm. D. Only cytoplasmic irradiation induces autophagy. SAE cells were irradiated with 5 α-particles through nucleus or cytoplasm. Cells were harvested 4 hr after irradiation and LC3B positive ratio were evaluated (n=3). E. Free radical scavenger attenuates autophagy induced by cytoplasmic irradiation. SAE cells were pre-treated with 0.5% DMSO or N-acetyl cysteine (NAC) for 30 min or 24 hr, respectively. LC3B positive ratio were evaluated 4 hr after cytoplasmic irradiation. CI, cytoplasmic irradiation. CTCF, corrected total cell fluorescence. Error bar ± S.D. * p

    Journal: Mutation research

    Article Title: Targeted Cytoplasmic Irradiation and Autophagy

    doi: 10.1016/j.mrfmmm.2017.02.004

    Figure Lengend Snippet: Targeted cytoplasmic irradiation induces autophagy in SAE cells A. LC3B puncta formation increases in SAE cells after cytoplasmic irradiation. SAE cells were treated with 5 α-particles 8 μm away from the cell nucleus along the major axis. Cells were incubated for 4 hr (left) or indicated time (right) before fixation and immunofluorescence staining for LC3B (green). Nuclei were visualized by DAPI (blue). LC3B positive ratio was quantified by counting cells with LC3B puncta based on total number of cells per dish in each time point (n=3). Scale bar = 10 μm. B. Autophagic flux assay proves the increase of autophagosomal synthesis. SAE cells were pre-treated with DMSO as control or 100 nM Bafilomycin A1 (Baf) for 1 hr before cytoplasmic irradiation. LC3B positive ratio were determined as described in Figure 1A. Scale bar = 10 μm. C. p62 expression reduces after cytoplasmic irradiation. SAE cells were cytoplasmic irradiated and incubated for 20 hr before fixation and immunofluorescence staining for p62 (green). Nuclei were visualized by DAPI (blue). Protein expression level was calculated using total cell fluorescence as described in Materials and Methods. Scale bar = 10 μm. D. Only cytoplasmic irradiation induces autophagy. SAE cells were irradiated with 5 α-particles through nucleus or cytoplasm. Cells were harvested 4 hr after irradiation and LC3B positive ratio were evaluated (n=3). E. Free radical scavenger attenuates autophagy induced by cytoplasmic irradiation. SAE cells were pre-treated with 0.5% DMSO or N-acetyl cysteine (NAC) for 30 min or 24 hr, respectively. LC3B positive ratio were evaluated 4 hr after cytoplasmic irradiation. CI, cytoplasmic irradiation. CTCF, corrected total cell fluorescence. Error bar ± S.D. * p

    Article Snippet: Anti-p62, phospho-ERK (T202/Y204), phospho-p70 S6 kinase (T389), phospho-AMPKα (T172), phospho-ATK (S473) and phospho-AKT (T308) antibodies were purchased from Cell Signaling.

    Techniques: Irradiation, Incubation, Immunofluorescence, Staining, Flux Assay, Expressing, Fluorescence

    Tigecycline induces autophagy of CML cells by downregulating the PI3K-ATK-mTOR signaling pathway. ( a ) Autophagic vacuoles were measured by transmission electron microscopy. Upper panel: autophagic vacuoles in CML cells with and without tigecycline treatment. Lower panel: amplified image of autophagic vacuoles in tigecycline-treated CML cells. ( b ) Confocal microscopy analysis of autophagy. Blue spots indicate nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Green spots indicate autophagic vacuoles stained with LC3B dye. ( c ) Western blot analysis to evaluate the levels of autophagy related protein P62 and LC3B, and mTOR and its upstream regulator AKT, and downstream sensors P70S6 and 4E-BP1

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inhibition of autophagy enhances the selective anti-cancer activity of tigecycline to overcome drug resistance in the treatment of chronic myeloid leukemia

    doi: 10.1186/s13046-017-0512-6

    Figure Lengend Snippet: Tigecycline induces autophagy of CML cells by downregulating the PI3K-ATK-mTOR signaling pathway. ( a ) Autophagic vacuoles were measured by transmission electron microscopy. Upper panel: autophagic vacuoles in CML cells with and without tigecycline treatment. Lower panel: amplified image of autophagic vacuoles in tigecycline-treated CML cells. ( b ) Confocal microscopy analysis of autophagy. Blue spots indicate nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). Green spots indicate autophagic vacuoles stained with LC3B dye. ( c ) Western blot analysis to evaluate the levels of autophagy related protein P62 and LC3B, and mTOR and its upstream regulator AKT, and downstream sensors P70S6 and 4E-BP1

    Article Snippet: The western blotting analysis was conducted using the following primary antibodies: anti-cytochrome c (11940), anti-cleaved-caspase-9 (32539), anti-cleaved-caspase-3 (13847), anti-LC3B (51520), and anti-p62 (91526), which were obtained from Abcam (Cambridge, UK); and p-AKT (Ser473, 4060), p-AKT (Thr308, 13038), AKT (9272), p-mTOR (Ser2448, 5536), p-P70S6K (Thr389, 9234), p-4E-BP1 (Thr37/46, 2855), and anti-ATG5 (12994), which were obtained from Cell Signaling Technology; and anti-Cox-1(19998), anti-Cox-2(19999) and anti-Cox-4 (69359), which were obtained from Santa Cruz Biotechnology (California, USA).

    Techniques: Transmission Assay, Electron Microscopy, Amplification, Confocal Microscopy, Staining, Western Blot

    Autophagy promotion is involved in the anti‐AML effect of matrine in vivo . ( A ) Experimental protocol was used to assess the anti‐AML effect of matrine on C57BL/6 mice. ( B ) After treated with matrine, chloroquine (CQ) or saline (Veh), IHC staining of SQSTM1/P62 and LC3 II in spleen and bone were observed by microscopy and representative images were showed. The scale bars are 50 μm. ( C ) IOD of SQSTM1/P62 and LC3 II of spleen and bone were showed in bar charts. ( D ) Wright's staining of peripheral blood and bone marrow was performed to observe immature leucocyte cells by microscopy, and representative images are showed. The scale bars are 10 μm, and arrows indicate immature leucocyte cells. ( E ) The percentage of immature leucocyte cells in peripheral blood and bone marrow was shown. ( F ) The spleen weight of mice was evaluated after killed. ( G ) The levels of SQSTM1/p62, LC3 II and cleaved caspase‐3 in bone marrow mononuclear cells were determined using Western blot analysis. ( H ) Kaplan–Meier curves for survival were assessed for 10 mice per group, and the long‐rank test was used to evaluate the statistical differences in survival. Results were expressed as mean ± S.E.M., and images shown were representatives of at least three independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Matrine induces Akt/mTOR signalling inhibition‐mediated autophagy and apoptosis in acute myeloid leukaemia cells

    doi: 10.1111/jcmm.13049

    Figure Lengend Snippet: Autophagy promotion is involved in the anti‐AML effect of matrine in vivo . ( A ) Experimental protocol was used to assess the anti‐AML effect of matrine on C57BL/6 mice. ( B ) After treated with matrine, chloroquine (CQ) or saline (Veh), IHC staining of SQSTM1/P62 and LC3 II in spleen and bone were observed by microscopy and representative images were showed. The scale bars are 50 μm. ( C ) IOD of SQSTM1/P62 and LC3 II of spleen and bone were showed in bar charts. ( D ) Wright's staining of peripheral blood and bone marrow was performed to observe immature leucocyte cells by microscopy, and representative images are showed. The scale bars are 10 μm, and arrows indicate immature leucocyte cells. ( E ) The percentage of immature leucocyte cells in peripheral blood and bone marrow was shown. ( F ) The spleen weight of mice was evaluated after killed. ( G ) The levels of SQSTM1/p62, LC3 II and cleaved caspase‐3 in bone marrow mononuclear cells were determined using Western blot analysis. ( H ) Kaplan–Meier curves for survival were assessed for 10 mice per group, and the long‐rank test was used to evaluate the statistical differences in survival. Results were expressed as mean ± S.E.M., and images shown were representatives of at least three independent experiments. * P

    Article Snippet: Firstly, we investigated the levels of SQSTM1/p62 and LC3 II, which are two key proteins in autophagy regulation after treatment with matrine for 24 hrs.

    Techniques: In Vivo, Mouse Assay, Immunohistochemistry, Staining, Microscopy, Western Blot

    Matrine induces autophagy in AML cells. ( A ) HL‐60, THP‐1, C1498 and primary AML cells were treated with 0, 0.25, 0.75, 1, 1.5, 2, 3 g/l matrine for 24 hrs, and the autophagy markers LC3 II and SQSTM1/p62 were detected by Western blot analysis. ( B ‐ C ) AML cells were treated with 1.5 g/l matrine in the presence or absence of 10 nM bafilomycin A1 (Baf A1) or 20 nM rapamycin (Rapa) for 24 hrs, and the levels of LC3 II and SQSTM1/p62 were assessed by Western blot analysis. ( D ) After treatment with 1.5 g/l matrine in the presence or absence of 10 nM Baf A1 or 20 nM rapamycin or 10 μM z‐VAD‐FMK (zVAD) for 24 hrs, the cell viability was measured by CCK‐8 assay. Results were expressed as mean ± S.E.M. representing at least three independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Matrine induces Akt/mTOR signalling inhibition‐mediated autophagy and apoptosis in acute myeloid leukaemia cells

    doi: 10.1111/jcmm.13049

    Figure Lengend Snippet: Matrine induces autophagy in AML cells. ( A ) HL‐60, THP‐1, C1498 and primary AML cells were treated with 0, 0.25, 0.75, 1, 1.5, 2, 3 g/l matrine for 24 hrs, and the autophagy markers LC3 II and SQSTM1/p62 were detected by Western blot analysis. ( B ‐ C ) AML cells were treated with 1.5 g/l matrine in the presence or absence of 10 nM bafilomycin A1 (Baf A1) or 20 nM rapamycin (Rapa) for 24 hrs, and the levels of LC3 II and SQSTM1/p62 were assessed by Western blot analysis. ( D ) After treatment with 1.5 g/l matrine in the presence or absence of 10 nM Baf A1 or 20 nM rapamycin or 10 μM z‐VAD‐FMK (zVAD) for 24 hrs, the cell viability was measured by CCK‐8 assay. Results were expressed as mean ± S.E.M. representing at least three independent experiments. * P

    Article Snippet: Firstly, we investigated the levels of SQSTM1/p62 and LC3 II, which are two key proteins in autophagy regulation after treatment with matrine for 24 hrs.

    Techniques: Western Blot, CCK-8 Assay

    H 2 S pretreatment suppressed TBI-induced the increase of Vps34 and P62. Sample immunoblots probed for Vps34, P62 and GAPDH are showed above. The bar chart below demonstrates the ratio of Vps34 and P62 relative to GAPDH. TBI-induced up-regulation of Vps34 and down-regulation of P62 were inhibited by H 2 S in the cortex (A) and hippocampus (B). Optical densities of the protein bands were quantitatively analyzed with Sigma Scan Pro 5 and normalized with loading control GAPDH. Semiquantitative analysis (relative optical density) of the intensity of staining of Vps34 to GAPDH in the cortex (C) and hippocampus (D). Semiquantitative analysis (relative optical density) of the intensity of staining of P62 to GAPDH in the cortex (E) and hippocampus (F). The data are means ± SEM (n = 3, *P

    Journal: PLoS ONE

    Article Title: Hydrogen Sulfide Offers Neuroprotection on Traumatic Brain Injury in Parallel with Reduced Apoptosis and Autophagy in Mice

    doi: 10.1371/journal.pone.0087241

    Figure Lengend Snippet: H 2 S pretreatment suppressed TBI-induced the increase of Vps34 and P62. Sample immunoblots probed for Vps34, P62 and GAPDH are showed above. The bar chart below demonstrates the ratio of Vps34 and P62 relative to GAPDH. TBI-induced up-regulation of Vps34 and down-regulation of P62 were inhibited by H 2 S in the cortex (A) and hippocampus (B). Optical densities of the protein bands were quantitatively analyzed with Sigma Scan Pro 5 and normalized with loading control GAPDH. Semiquantitative analysis (relative optical density) of the intensity of staining of Vps34 to GAPDH in the cortex (C) and hippocampus (D). Semiquantitative analysis (relative optical density) of the intensity of staining of P62 to GAPDH in the cortex (E) and hippocampus (F). The data are means ± SEM (n = 3, *P

    Article Snippet: The separated proteins were transferred to a polyvinylidine difluoride membrane (Millipore) by a transfer apparatus at 350 mA for 1.5 h. The membrane was then blocked with 5% nonfat milk and incubated with primary antibody against procaspase-3 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3 (1:500; Bioword Technology, Minneapolis, MN, USA), Bcl-2 (1:1000; Bioword Technology, Minneapolis, MN, USA), Beclin-1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Vps34 (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3B (1:3000, Abcam, Cambridge, MA, USA), p62 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GAPDH (1:10000; Bioword Technology, Minneapolis, MN, USA).

    Techniques: Western Blot, Staining

    TFEB silencing counteracts trehalose-induced expression of autophagic genes. ( a-n ) NSC34 cells were transfected with Tfeb or non-targeting (as control) siRNAs and treated with 100 mM glucose or trehalose. ( a ) WB analysis was performed, and the bar graphs (b-c) represent the mean relative optical density of SQSTM1/p62 and MAP1LC3B-II:MAP1LC3B-I protein expression levels, respectively, performed with n = 3 independent samples. LC3-II:LC3-I ratio was calculated by densitometric analysis of both bands. (*** pÂÂ

    Journal: Autophagy

    Article Title: Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration

    doi: 10.1080/15548627.2018.1535292

    Figure Lengend Snippet: TFEB silencing counteracts trehalose-induced expression of autophagic genes. ( a-n ) NSC34 cells were transfected with Tfeb or non-targeting (as control) siRNAs and treated with 100 mM glucose or trehalose. ( a ) WB analysis was performed, and the bar graphs (b-c) represent the mean relative optical density of SQSTM1/p62 and MAP1LC3B-II:MAP1LC3B-I protein expression levels, respectively, performed with n = 3 independent samples. LC3-II:LC3-I ratio was calculated by densitometric analysis of both bands. (*** pÂÂ

    Article Snippet: The membranes obtained by WB or FRA were immunoblotted using the following antibodies: anti-SQSTM1/p62 (Abcam, ab91526; 1:2,000); anti-LC3A/B (Sigma-Aldrich, L8918, 1:2,000); anti-TFEB (Bethyl Laboratories, A303-673A, 1:4,000), anti-phospho-TFEB (Ser142; Merck-Millipore, ABE1971; 1:3,000), anti-GAPDH (FL-335; Santa-Cruz Biotechnology, sc-25778; 1:3,000), anti-AR (H280; Santa Cruz Biotechnology, sc-13062; 1:1,000), anti-histone H3 (Abcam, ab1791; 1:40,000), anti-GFP (Abcam, AB1218; 1:2,000).

    Techniques: Expressing, Transfection, Western Blot

    Melibiose and lactulose effects are mediated by PPP3CB: induction of ALP gene expression and LMP. ( a-b ) WB analysis of cytoplasmic (c) and nuclear extracts (N) of NSC34 cells untreated (control) or treated with 100 mM melibiose ( a ) or 100 mM lactulose ( b) in the absence or presence of 10 μM CsA for 1 h. GAPDH and histone H3 were used as internal loading controls for cytoplasmic and nuclear fractions, respectively. ( c-h ) RT-qPCR on NSC34 cells untreated (control) or treated with 100 mM trehalose, 100 mM melibiose or 100 mM lactulose for 48 h. The relative fold difference of mRNA expression was determined using untreated samples as internal control. Data are means ± SD of 4 independent samples. RT-qPCR for the following mRNA: Tfeb ( c ); Zkscan3 ( d ); Sqstm1 /p62 ( e ); Map1Lc3b ( f ); Hspb8 ( g ); Bag3 ( h) . Bar graphs represent the relative fold induction of these genes (*pÂÂ

    Journal: Autophagy

    Article Title: Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration

    doi: 10.1080/15548627.2018.1535292

    Figure Lengend Snippet: Melibiose and lactulose effects are mediated by PPP3CB: induction of ALP gene expression and LMP. ( a-b ) WB analysis of cytoplasmic (c) and nuclear extracts (N) of NSC34 cells untreated (control) or treated with 100 mM melibiose ( a ) or 100 mM lactulose ( b) in the absence or presence of 10 μM CsA for 1 h. GAPDH and histone H3 were used as internal loading controls for cytoplasmic and nuclear fractions, respectively. ( c-h ) RT-qPCR on NSC34 cells untreated (control) or treated with 100 mM trehalose, 100 mM melibiose or 100 mM lactulose for 48 h. The relative fold difference of mRNA expression was determined using untreated samples as internal control. Data are means ± SD of 4 independent samples. RT-qPCR for the following mRNA: Tfeb ( c ); Zkscan3 ( d ); Sqstm1 /p62 ( e ); Map1Lc3b ( f ); Hspb8 ( g ); Bag3 ( h) . Bar graphs represent the relative fold induction of these genes (*pÂÂ

    Article Snippet: The membranes obtained by WB or FRA were immunoblotted using the following antibodies: anti-SQSTM1/p62 (Abcam, ab91526; 1:2,000); anti-LC3A/B (Sigma-Aldrich, L8918, 1:2,000); anti-TFEB (Bethyl Laboratories, A303-673A, 1:4,000), anti-phospho-TFEB (Ser142; Merck-Millipore, ABE1971; 1:3,000), anti-GAPDH (FL-335; Santa-Cruz Biotechnology, sc-25778; 1:3,000), anti-AR (H280; Santa Cruz Biotechnology, sc-13062; 1:1,000), anti-histone H3 (Abcam, ab1791; 1:40,000), anti-GFP (Abcam, AB1218; 1:2,000).

    Techniques: ALP Assay, Expressing, Western Blot, Quantitative RT-PCR

    CIP2A and c-Myc are degraded in an autophagy-dependent manner. (A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0–24 h. (B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments. (C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control. (D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0–5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA. (E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm. (F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control. (G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (

    Journal: The Journal of Cell Biology

    Article Title: CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation

    doi: 10.1083/jcb.201304012

    Figure Lengend Snippet: CIP2A and c-Myc are degraded in an autophagy-dependent manner. (A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0–24 h. (B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments. (C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control. (D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0–5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA. (E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm. (F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control. (G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (

    Article Snippet: Immunodetection The primary antibodies used included rabbit antibodies against P-Thr308-Akt (4056), P-Ser473-Akt (4060), P-Thr1462-TSC2 (3611), P-Ser2448-mTOR (2971), pan-S6K1 (2708), P-Thr389-S6K1 (9234), P-Thr421/Ser424-S6K1 (9204), P-Thr37/46-4E-BP1 (236B4), 4E-BP1 (9644), S6 (4857), and P-Ser235/Ser236-S6 (4856) obtained from Cell Signaling Technology, P-S318-ATG13 (600–401-c49) obtained from Rockland Immunochemicals, Inc., p62/SQSTM1 (ab31545) obtained from Abcam, polyubiquitin (U5379) purchased from Sigma-Aldrich, and PR65 (SC-15355) purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Staining, Immunoprecipitation, Negative Control

    G-TPP activity is conserved in primary fibroblasts ( A , C ) Fibroblasts were treated with 15 µM G-TPP for the indicated time points. Cells were harvested and western blots were probed with antibodies against (A) PINK1, pS65-Ub and total Ub or (C) autophagy adapter proteins. GAPDH and Vinculin served as loading control. G-TPP treatment led to PINK1 stabilization and pS65-Ub induction in primary skin fibroblasts. p62 levels were induced upon G-TPP treatment, while other adapters seemed decreased. ( B , D ) Human fibroblasts were treated with 15 µM G-TPP for 16 h and fixed and stained with antibodies against (B) pS65-Ub (green) or (D) the autophagy adapters NBR1, NDP52, p62, OPTN and TAX1BP1 (green). Mitochondria were stained with antibodies against TOM20 (red), nuclei were visualized with Hoechst (blue). Scale bars indicate 10 µM. A magnified image of the boxed region, the fluorescence profile along the arrow and the Pearson’s correlation coefficient of adapter protein and mitochondrial stainingare shown to the right. Shown is the mean ± SEM of at least five randomly selected images (unpaired, two-sided t -test, *** p

    Journal: Oncotarget

    Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

    doi: 10.18632/oncotarget.22287

    Figure Lengend Snippet: G-TPP activity is conserved in primary fibroblasts ( A , C ) Fibroblasts were treated with 15 µM G-TPP for the indicated time points. Cells were harvested and western blots were probed with antibodies against (A) PINK1, pS65-Ub and total Ub or (C) autophagy adapter proteins. GAPDH and Vinculin served as loading control. G-TPP treatment led to PINK1 stabilization and pS65-Ub induction in primary skin fibroblasts. p62 levels were induced upon G-TPP treatment, while other adapters seemed decreased. ( B , D ) Human fibroblasts were treated with 15 µM G-TPP for 16 h and fixed and stained with antibodies against (B) pS65-Ub (green) or (D) the autophagy adapters NBR1, NDP52, p62, OPTN and TAX1BP1 (green). Mitochondria were stained with antibodies against TOM20 (red), nuclei were visualized with Hoechst (blue). Scale bars indicate 10 µM. A magnified image of the boxed region, the fluorescence profile along the arrow and the Pearson’s correlation coefficient of adapter protein and mitochondrial stainingare shown to the right. Shown is the mean ± SEM of at least five randomly selected images (unpaired, two-sided t -test, *** p

    Article Snippet: Antibodies The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

    Techniques: Activity Assay, Western Blot, Staining, Fluorescence

    G-TPP leads to recruitment of autophagy adapters and degradation of mitochondria ( A ) HeLa cells stably expressing untagged Parkin were treated with 10 µM G-TPP for 8 h. Western blots were prepared from cell lysates and probed with antibodies against LC3, phospho-TBK1 (Ser172) and TBK1. GAPDH was used as a loading control. Upon 8 h the levels of LC3-I and LC3-II were both increased. At 8 h after treatment with G-TPP but not at 4 or 24 h, TBK1 was phosphorylated. ( B ) HeLa cells stably expressing EGFP-Parkin were treated with 10 µM G-TPP and fixed 8 h after treatment. Cells were stained with antibodies against the autophagy adapter proteins NBR1, NDP52, OPTN, p62, and TAX1BP1 (red). Mitochondria were counterstained with TOM20 antibodies (cyan), nuclei with Hoechst (blue). EGFP-Parkin epifluorescence is shown in green. Scale bar corresponds to 10 µM. ( C ) HeLa cells stably expressing EGFP-Parkin and the reporter protein mitoKeima were treated with 10 µM CCCP or G-TPP and imaged over time. The ratio of ‘neutral’ mitoKeima to ‘acidic’ mitoKeima was calculated as readout for mitophagy. Parkin translocation was monitored at the same time. Values for Parkin translocation and mitophagy were normalized to 12 h treatment with 10 µM CCCP as positive control and DMSO as negative control (two-way ANOVA with Tukey’s post-hoc test, ** p

    Journal: Oncotarget

    Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy

    doi: 10.18632/oncotarget.22287

    Figure Lengend Snippet: G-TPP leads to recruitment of autophagy adapters and degradation of mitochondria ( A ) HeLa cells stably expressing untagged Parkin were treated with 10 µM G-TPP for 8 h. Western blots were prepared from cell lysates and probed with antibodies against LC3, phospho-TBK1 (Ser172) and TBK1. GAPDH was used as a loading control. Upon 8 h the levels of LC3-I and LC3-II were both increased. At 8 h after treatment with G-TPP but not at 4 or 24 h, TBK1 was phosphorylated. ( B ) HeLa cells stably expressing EGFP-Parkin were treated with 10 µM G-TPP and fixed 8 h after treatment. Cells were stained with antibodies against the autophagy adapter proteins NBR1, NDP52, OPTN, p62, and TAX1BP1 (red). Mitochondria were counterstained with TOM20 antibodies (cyan), nuclei with Hoechst (blue). EGFP-Parkin epifluorescence is shown in green. Scale bar corresponds to 10 µM. ( C ) HeLa cells stably expressing EGFP-Parkin and the reporter protein mitoKeima were treated with 10 µM CCCP or G-TPP and imaged over time. The ratio of ‘neutral’ mitoKeima to ‘acidic’ mitoKeima was calculated as readout for mitophagy. Parkin translocation was monitored at the same time. Values for Parkin translocation and mitophagy were normalized to 12 h treatment with 10 µM CCCP as positive control and DMSO as negative control (two-way ANOVA with Tukey’s post-hoc test, ** p

    Article Snippet: Antibodies The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).

    Techniques: Stable Transfection, Expressing, Western Blot, Staining, Translocation Assay, Positive Control, Negative Control

    The antiviral effect of ANKS4B was exerted through inhibiting the autophagy. (A) Co-IP assay. A549 and 293T cells were co-transfected with plasmids expressing ANKS4B-FLAG protein and GRP78. Whole cell extracts were prepared for Co-IP assay using anti-FLAG. (B) Control, ANKS4B-KO1, ANKS4B-KO2, and ANKS4B-RES (R) Huh7 cells were infected with ZIKV (MOI 3). Cell lysates were prepared at 24 h p.i for western blot to detect the expression levels of ZIKV E protein, LC3B, and p62. (C–F) Autophagy inhibitor assay. Control, ANKS4B-KO1, ANKS4B-KO2, and ANKS4B-RES cells were treated with DMSO or chloroquine (CQ, 50 μM) or 3-methyladenine (3-MA, 5 mM) after ZIKV inoculation. At 24 h p.i., cells and supernatants were harvested for western blot (C,E) or plaque assay (D,F) . Levels of p62, ZIKV E, and GAPDH were measured. All the data were shown as means ± S.D. (error bars) from at least three independent experiments. NS, not significant. * p

    Journal: Frontiers in Microbiology

    Article Title: ANKS4B Restricts Replication of Zika Virus by Downregulating the Autophagy

    doi: 10.3389/fmicb.2020.01745

    Figure Lengend Snippet: The antiviral effect of ANKS4B was exerted through inhibiting the autophagy. (A) Co-IP assay. A549 and 293T cells were co-transfected with plasmids expressing ANKS4B-FLAG protein and GRP78. Whole cell extracts were prepared for Co-IP assay using anti-FLAG. (B) Control, ANKS4B-KO1, ANKS4B-KO2, and ANKS4B-RES (R) Huh7 cells were infected with ZIKV (MOI 3). Cell lysates were prepared at 24 h p.i for western blot to detect the expression levels of ZIKV E protein, LC3B, and p62. (C–F) Autophagy inhibitor assay. Control, ANKS4B-KO1, ANKS4B-KO2, and ANKS4B-RES cells were treated with DMSO or chloroquine (CQ, 50 μM) or 3-methyladenine (3-MA, 5 mM) after ZIKV inoculation. At 24 h p.i., cells and supernatants were harvested for western blot (C,E) or plaque assay (D,F) . Levels of p62, ZIKV E, and GAPDH were measured. All the data were shown as means ± S.D. (error bars) from at least three independent experiments. NS, not significant. * p

    Article Snippet: The primary antibodies included: anti-ZIKV envelope (E) (GeneTex, GTX 133314), anti-p62 (Santa Cruz, sc-28359), anti-GRP78 (Proteintech, 11587-1-AP), anti-Calnexin (Proteintech, 66903-1-lg), anti-FLAG (MBL, PM020), and anti-anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, 10494-1-AP).

    Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Infection, Western Blot, Plaque Assay

    Microglia-engulfed α-synuclein is sequestered by autophagosomes and degraded by autophagy. (a, b, c) Cultured primary microglia from WT mice (a and left panels of b) and GFP-LC3-transgenic mice (right panels of b) were treated 250nM h α-Syn protein for indicated time and stained with antibodies against human α-synuclein (MJFR1 clone, a and b), ubiquitin ( a ), or p62 ( b ). The number of h α-Syn/ubiquitin-positive puncta was quantified ( c ). In (a), empty arrowheads indicate a portion of α-synuclein colocalized with ubiquitin-positive puncta and disappear in 24 hours. Arrows represent a portion of α-synuclein not colocalized with ubiquitin. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. Scale bar, 10 µm; 2 µm in magnified boxes. (d) At 6-weeks post-AAV- h α-Syn inoculation into GFP-LC3 mice, brain slices were fixed and stained with antibodies against human α-synuclein, GFP, and Iba-1 (left panel). 3D surface-rendering images of each protein in microglia were made with Imaris software (Bitplane, right panel). Scale bar, 10µm; 1µm in the magnified box. (e) Ultrastructure of puncta from 250nM h α-Syn protein-treated cells for 6 hours was observed by an electron microscope. Arrows indicate double-membrane structures. Scale bar, 500 nm in the left panel; 100 nm in the right panel. (f, g) Microglia were treated with 250nM h α-Syn protein for 6 hours (f) or 3 hours (g) first and added SAR405 ( f ) or Torin ( g ) was added. The number of h α-Syn/ubiquitin-positive puncta was quantified in the lower panel. p values were calculated by One-way ANOVA with Newman– Keuls post hoc test (f) and Unpaired two-tailed Student’s t-test (g). (h, i) Microglia cultured either from Atg7 flox/flox mice (left panel) from Atg14 flox/flox mice (right panel) with or without Cx3cr1 Cre expression were assayed for W.B using antibodies against ATG7 or ATG14, p62, and LC3 I/II ( h ). Cells were treated with 250nM h α-Syn protein for 24 hours and the number of h α-Syn/ubiquitin-positive puncta was quantified ( i ). p values were calculated by Mann-Whitney U test. Data are representative of at least three independent experiments.

    Journal: bioRxiv

    Article Title: Microglia Clear Neuron-released α-Synuclein via Selective Autophagy and Prevent Neurodegeneration

    doi: 10.1101/2019.12.11.872812

    Figure Lengend Snippet: Microglia-engulfed α-synuclein is sequestered by autophagosomes and degraded by autophagy. (a, b, c) Cultured primary microglia from WT mice (a and left panels of b) and GFP-LC3-transgenic mice (right panels of b) were treated 250nM h α-Syn protein for indicated time and stained with antibodies against human α-synuclein (MJFR1 clone, a and b), ubiquitin ( a ), or p62 ( b ). The number of h α-Syn/ubiquitin-positive puncta was quantified ( c ). In (a), empty arrowheads indicate a portion of α-synuclein colocalized with ubiquitin-positive puncta and disappear in 24 hours. Arrows represent a portion of α-synuclein not colocalized with ubiquitin. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. Scale bar, 10 µm; 2 µm in magnified boxes. (d) At 6-weeks post-AAV- h α-Syn inoculation into GFP-LC3 mice, brain slices were fixed and stained with antibodies against human α-synuclein, GFP, and Iba-1 (left panel). 3D surface-rendering images of each protein in microglia were made with Imaris software (Bitplane, right panel). Scale bar, 10µm; 1µm in the magnified box. (e) Ultrastructure of puncta from 250nM h α-Syn protein-treated cells for 6 hours was observed by an electron microscope. Arrows indicate double-membrane structures. Scale bar, 500 nm in the left panel; 100 nm in the right panel. (f, g) Microglia were treated with 250nM h α-Syn protein for 6 hours (f) or 3 hours (g) first and added SAR405 ( f ) or Torin ( g ) was added. The number of h α-Syn/ubiquitin-positive puncta was quantified in the lower panel. p values were calculated by One-way ANOVA with Newman– Keuls post hoc test (f) and Unpaired two-tailed Student’s t-test (g). (h, i) Microglia cultured either from Atg7 flox/flox mice (left panel) from Atg14 flox/flox mice (right panel) with or without Cx3cr1 Cre expression were assayed for W.B using antibodies against ATG7 or ATG14, p62, and LC3 I/II ( h ). Cells were treated with 250nM h α-Syn protein for 24 hours and the number of h α-Syn/ubiquitin-positive puncta was quantified ( i ). p values were calculated by Mann-Whitney U test. Data are representative of at least three independent experiments.

    Article Snippet: For cultured primary microglia, cells were fixed with 4% paraformaldehyde at 4□ for 15 minutes and incubated with microwave-boiled antigen retrieval solution (#CTS013, R & D System) for 5 min. After washing with PBS, cells were incubated with blocking/permeabilization buffer (1% BSA and 0.1%Triton X-100 in PBS) at RT for 30 minutes and incubated with primary antibodies including EEA1 (610457, BD Bioscience), α-synuclein (clone MJFR1, ab138501, Abcam; clone 2f12, MABN1817, Millipore; clone 42, 610787 BD Bioscience; clone 204, #2647, Cell Signaling), p62 (GP62-C, Progen, Germany), ubiquitin (clone P4D1, sc-8017, Santa Cruz Biotechnology), and TFEB (A303-673A, Bethyl Laboratories Inc, TX) at 4□ overnight followed by secondary antibodies.

    Techniques: Cell Culture, Mouse Assay, Transgenic Assay, Staining, Software, Microscopy, Two Tailed Test, Expressing, MANN-WHITNEY

    NF-κB mediates α-synuclein - TLR4 signaling and p62 induction in microglia. (a) WT microglia were treated with 250nM h α-Syn protein for the indicated time and assayed for W.B using antibodies against p-NF-κB (S536), IkB, p-p38 (T180/Y182), p38, p-ERK1/2 (T202/Y204), ERK1/2, p-JNK (T183/Y185), JNK, and human α-synuclein. The levels of proteins were quantified in the right panel. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (b, c) Cells were pretreated with ML120B, an IKK-2 inhibitor, or SB203580 and SB202190, two different p38 inhibitors, for indicated concentrations before 250nM h α-Syn protein treatment, and assayed for W.B ( b ) or immunostaining ( c ). The levels of proteins were quantified in the right panel. The number of h α-Syn/ubiquitin-positive puncta was counted and quantified (c). p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (d-f) Microglia obtained from WT mice and Tlr4 -KO mice were treated with h α-Syn protein for 3 hours with indicated concentration without ( d ) or with ( e, f ) TLR2-blocking antibody (T2.5), and assayed for RT-qPCR using primers for Il-1b (left panel) and Tnf (right panel). p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test (wt vs ko in d), One-way ANOVA with Newman–Keuls post hoc test (non-treated vs. treated in d, e), and Unpaired two-tailed Student’s t-test (f). (g-i) HEK293T cells were transfected with a luciferase vector that expresses luciferase under the promoter containing NF-κB-binding elements, and various TLRs. Then, cells were treated h α-Syn protein with indicated concentration (g, h, i), 50ng/ml LPS (i), or Pam 3 CSK 4 (i) for 24 hours without (g, h) or with (i) 10µg/ml TLR2-blocking antibody (T2.5) and control IgG. p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

    Journal: bioRxiv

    Article Title: Microglia Clear Neuron-released α-Synuclein via Selective Autophagy and Prevent Neurodegeneration

    doi: 10.1101/2019.12.11.872812

    Figure Lengend Snippet: NF-κB mediates α-synuclein - TLR4 signaling and p62 induction in microglia. (a) WT microglia were treated with 250nM h α-Syn protein for the indicated time and assayed for W.B using antibodies against p-NF-κB (S536), IkB, p-p38 (T180/Y182), p38, p-ERK1/2 (T202/Y204), ERK1/2, p-JNK (T183/Y185), JNK, and human α-synuclein. The levels of proteins were quantified in the right panel. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (b, c) Cells were pretreated with ML120B, an IKK-2 inhibitor, or SB203580 and SB202190, two different p38 inhibitors, for indicated concentrations before 250nM h α-Syn protein treatment, and assayed for W.B ( b ) or immunostaining ( c ). The levels of proteins were quantified in the right panel. The number of h α-Syn/ubiquitin-positive puncta was counted and quantified (c). p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (d-f) Microglia obtained from WT mice and Tlr4 -KO mice were treated with h α-Syn protein for 3 hours with indicated concentration without ( d ) or with ( e, f ) TLR2-blocking antibody (T2.5), and assayed for RT-qPCR using primers for Il-1b (left panel) and Tnf (right panel). p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test (wt vs ko in d), One-way ANOVA with Newman–Keuls post hoc test (non-treated vs. treated in d, e), and Unpaired two-tailed Student’s t-test (f). (g-i) HEK293T cells were transfected with a luciferase vector that expresses luciferase under the promoter containing NF-κB-binding elements, and various TLRs. Then, cells were treated h α-Syn protein with indicated concentration (g, h, i), 50ng/ml LPS (i), or Pam 3 CSK 4 (i) for 24 hours without (g, h) or with (i) 10µg/ml TLR2-blocking antibody (T2.5) and control IgG. p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

    Article Snippet: For cultured primary microglia, cells were fixed with 4% paraformaldehyde at 4□ for 15 minutes and incubated with microwave-boiled antigen retrieval solution (#CTS013, R & D System) for 5 min. After washing with PBS, cells were incubated with blocking/permeabilization buffer (1% BSA and 0.1%Triton X-100 in PBS) at RT for 30 minutes and incubated with primary antibodies including EEA1 (610457, BD Bioscience), α-synuclein (clone MJFR1, ab138501, Abcam; clone 2f12, MABN1817, Millipore; clone 42, 610787 BD Bioscience; clone 204, #2647, Cell Signaling), p62 (GP62-C, Progen, Germany), ubiquitin (clone P4D1, sc-8017, Santa Cruz Biotechnology), and TFEB (A303-673A, Bethyl Laboratories Inc, TX) at 4□ overnight followed by secondary antibodies.

    Techniques: Immunostaining, Mouse Assay, Concentration Assay, Blocking Assay, Quantitative RT-PCR, Two Tailed Test, Transfection, Luciferase, Plasmid Preparation, Binding Assay

    Transcriptional upregulation of autophagy receptor p62 mediates microglial sequestration and degradation of α-synuclein. (a) Microglia were treated with 250nM h α-Syn protein for the indicated time and assayed for W.B using antibodies against p62 and human α-synuclein. The levels of p62 protein were quantified in the lower panel. p values were calculated by One-way ANOVA with Newman– Keuls post hoc test. (b) After treatment of 250nM h α-Syn protein for the indicated time, the levels of p62 mRNA were examined by RT-qPCR. p62 mRNA was normalized to Actin mRNA. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (c) The absence of p62 protein was confirmed in microglia obtained from p62 -KO mice compared to WT mice by W.B. (d, e) WT and p62 -KO microglia were treated with 250nM h α-Syn protein for 6 hours and stained with antibodies against human α-synuclein and ubiquitin ( d ). The number of h α-Syn/ubiquitin-positive puncta was quantified ( e ). p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test. Scale bar, 10 µm. (f-h) The number of p62 puncta was quantified in microglia at striatum of brains either from AAV- h α-Syn-injected mice (n=3, f, g ) or from h α-Syn-Tg mice (n=5, h ) compared to AAV-GFP-injected mice (n=3) and non-Tg mice (n=3), respectively. p values were calculated by Mann–Whitney U test. Scale bar, 10 µm. (i) At 6-weeks post-AAV- h α-Syn administration into Cx3cr1 CreER-IRES-Eyfp mice, brain slices were stained with antibodies against GFP/YFP, human α-synuclein, p62 (left panel), and ubiquitin (right panel). Scale bar, 10 µm; 1µm in the magnified box. (j) Brain sections from 10-months-old h α-syn-Tg mice were fixed and stained using antibodies against human α-synuclein, Iba-1, and p62. Scale bar, 10 µm; 1µm in the magnified box. (k, l, m) Human α-synuclein were immunoprecipitated from AAV - h α-Syn-injected brain ( k ), h α-Syn-Tg brain ( l ), and cultured microglia treated with 250nM h α-Syn protein for 6 hours ( m ). Arrows indicate the high-molecular-weight of p62. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

    Journal: bioRxiv

    Article Title: Microglia Clear Neuron-released α-Synuclein via Selective Autophagy and Prevent Neurodegeneration

    doi: 10.1101/2019.12.11.872812

    Figure Lengend Snippet: Transcriptional upregulation of autophagy receptor p62 mediates microglial sequestration and degradation of α-synuclein. (a) Microglia were treated with 250nM h α-Syn protein for the indicated time and assayed for W.B using antibodies against p62 and human α-synuclein. The levels of p62 protein were quantified in the lower panel. p values were calculated by One-way ANOVA with Newman– Keuls post hoc test. (b) After treatment of 250nM h α-Syn protein for the indicated time, the levels of p62 mRNA were examined by RT-qPCR. p62 mRNA was normalized to Actin mRNA. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (c) The absence of p62 protein was confirmed in microglia obtained from p62 -KO mice compared to WT mice by W.B. (d, e) WT and p62 -KO microglia were treated with 250nM h α-Syn protein for 6 hours and stained with antibodies against human α-synuclein and ubiquitin ( d ). The number of h α-Syn/ubiquitin-positive puncta was quantified ( e ). p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test. Scale bar, 10 µm. (f-h) The number of p62 puncta was quantified in microglia at striatum of brains either from AAV- h α-Syn-injected mice (n=3, f, g ) or from h α-Syn-Tg mice (n=5, h ) compared to AAV-GFP-injected mice (n=3) and non-Tg mice (n=3), respectively. p values were calculated by Mann–Whitney U test. Scale bar, 10 µm. (i) At 6-weeks post-AAV- h α-Syn administration into Cx3cr1 CreER-IRES-Eyfp mice, brain slices were stained with antibodies against GFP/YFP, human α-synuclein, p62 (left panel), and ubiquitin (right panel). Scale bar, 10 µm; 1µm in the magnified box. (j) Brain sections from 10-months-old h α-syn-Tg mice were fixed and stained using antibodies against human α-synuclein, Iba-1, and p62. Scale bar, 10 µm; 1µm in the magnified box. (k, l, m) Human α-synuclein were immunoprecipitated from AAV - h α-Syn-injected brain ( k ), h α-Syn-Tg brain ( l ), and cultured microglia treated with 250nM h α-Syn protein for 6 hours ( m ). Arrows indicate the high-molecular-weight of p62. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

    Article Snippet: For cultured primary microglia, cells were fixed with 4% paraformaldehyde at 4□ for 15 minutes and incubated with microwave-boiled antigen retrieval solution (#CTS013, R & D System) for 5 min. After washing with PBS, cells were incubated with blocking/permeabilization buffer (1% BSA and 0.1%Triton X-100 in PBS) at RT for 30 minutes and incubated with primary antibodies including EEA1 (610457, BD Bioscience), α-synuclein (clone MJFR1, ab138501, Abcam; clone 2f12, MABN1817, Millipore; clone 42, 610787 BD Bioscience; clone 204, #2647, Cell Signaling), p62 (GP62-C, Progen, Germany), ubiquitin (clone P4D1, sc-8017, Santa Cruz Biotechnology), and TFEB (A303-673A, Bethyl Laboratories Inc, TX) at 4□ overnight followed by secondary antibodies.

    Techniques: Quantitative RT-PCR, Mouse Assay, Staining, Injection, MANN-WHITNEY, Immunoprecipitation, Cell Culture, Molecular Weight

    α-synuclein-induced p62 upregulation requires TLR4 independently of TLR4 endocytosis. (a, b, c) Microglia obtained from WT mice and Tlr4 -KO mice were treated with h α-Syn protein and assayed for W.B ( a ), RT-qPCR ( b ) or for immunostaining ( c ). Band intensities were quantified in the right panel of a. The number of h α-Syn/ubiquitin-positive puncta was quantified (c). p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test. (d, e) Cells were pretreated with TAK242, a TLR4 signaling blocker, and treated with 250nM h α-Syn protein. The protein levels of p62 and the number of h α-Syn/ubiquitin-positive puncta were determined by W.B ( d ) and immunostaining ( e ), respectively. p values were calculated by Unpaired two-tailed Student’s t-test (d) and One-way ANOVA with Newman–Keuls post hoc test (e). (f, g) Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4 -KO mice injected with AAV- h α-Syn brains in ( f ). The number of p62 puncta was quantified in microglia at striatum of WT mice (n=5) and Tlr4 -KO mice (n=5) ( g ). p values were calculated by Mann–Whitney U test. Scale bar, 10 µm. (h) Microglia were treated with either 250nM h α-Syn protein (red bar) or 50ng/ml LPS (blue bar) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (i) After treating 250nM h α-Syn protein or 50ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. (j) After treating h α-Syn protein or LPS for 30 minutes to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. Mean fluorescence intensity of TLR4 receptor staining on the surface was quantified. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (k) Microglia were pretreated with Dynasore for indicated concentration and treated with 250nM h α-Syn protein for 3 hours. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

    Journal: bioRxiv

    Article Title: Microglia Clear Neuron-released α-Synuclein via Selective Autophagy and Prevent Neurodegeneration

    doi: 10.1101/2019.12.11.872812

    Figure Lengend Snippet: α-synuclein-induced p62 upregulation requires TLR4 independently of TLR4 endocytosis. (a, b, c) Microglia obtained from WT mice and Tlr4 -KO mice were treated with h α-Syn protein and assayed for W.B ( a ), RT-qPCR ( b ) or for immunostaining ( c ). Band intensities were quantified in the right panel of a. The number of h α-Syn/ubiquitin-positive puncta was quantified (c). p values were calculated by Two-way ANOVA with Bonferroni Post Hoc Test. (d, e) Cells were pretreated with TAK242, a TLR4 signaling blocker, and treated with 250nM h α-Syn protein. The protein levels of p62 and the number of h α-Syn/ubiquitin-positive puncta were determined by W.B ( d ) and immunostaining ( e ), respectively. p values were calculated by Unpaired two-tailed Student’s t-test (d) and One-way ANOVA with Newman–Keuls post hoc test (e). (f, g) Representative 3D reconstruction pictures of microglia containing p62 in WT and Tlr4 -KO mice injected with AAV- h α-Syn brains in ( f ). The number of p62 puncta was quantified in microglia at striatum of WT mice (n=5) and Tlr4 -KO mice (n=5) ( g ). p values were calculated by Mann–Whitney U test. Scale bar, 10 µm. (h) Microglia were treated with either 250nM h α-Syn protein (red bar) or 50ng/ml LPS (blue bar) for indicated time and assayed for RT-qPCR. Each mRNA level was normalized to Actin mRNA. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (i) After treating 250nM h α-Syn protein or 50ng/ml LPS for indicated time, cells were assayed for W.B using antibodies against p-IRF3 (S396), IRF3, and α-synuclein. (j) After treating h α-Syn protein or LPS for 30 minutes to microglia, the level of TLR4 receptor endocytosis was determined by detecting surface TLR4 using flow cytometry. Mean fluorescence intensity of TLR4 receptor staining on the surface was quantified. p values were calculated by One-way ANOVA with Newman–Keuls post hoc test. (k) Microglia were pretreated with Dynasore for indicated concentration and treated with 250nM h α-Syn protein for 3 hours. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

    Article Snippet: For cultured primary microglia, cells were fixed with 4% paraformaldehyde at 4□ for 15 minutes and incubated with microwave-boiled antigen retrieval solution (#CTS013, R & D System) for 5 min. After washing with PBS, cells were incubated with blocking/permeabilization buffer (1% BSA and 0.1%Triton X-100 in PBS) at RT for 30 minutes and incubated with primary antibodies including EEA1 (610457, BD Bioscience), α-synuclein (clone MJFR1, ab138501, Abcam; clone 2f12, MABN1817, Millipore; clone 42, 610787 BD Bioscience; clone 204, #2647, Cell Signaling), p62 (GP62-C, Progen, Germany), ubiquitin (clone P4D1, sc-8017, Santa Cruz Biotechnology), and TFEB (A303-673A, Bethyl Laboratories Inc, TX) at 4□ overnight followed by secondary antibodies.

    Techniques: Mouse Assay, Quantitative RT-PCR, Immunostaining, Two Tailed Test, Injection, MANN-WHITNEY, Flow Cytometry, Fluorescence, Staining, Concentration Assay

    Disruption of autophagy in microglia promotes accumulation of misfolded α-synuclein species in mouse brain. (a) Experimental plan to test the roles of microglia-specific Atg7 -deficiency in AAV- h α-Syn mice (b) After 6-weeks after AAV- h α-Syn, brains from Atg7 flox/flox mice (n=4) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox mice (n=3) were homogenized and fractionated into a detergent-soluble fraction and detergent-insoluble fraction and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP. Actin were used as loading controls. Arrow indicates the EYFP band. (c) Experimental plan to test the roles of microglia-specific Atg7 -deficiency in h α-Syn-Tg mice (d, e) After 7-months after tamoxifen, brain slices were fixed and stained with antibodies against human α-synuclein, p62, and Iba-1. Representative 3D reconstruction pictures of microglia containing p62 and human α-synuclein in the striatum of Atg7 flox/flox mice and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox mice bred with h α-Syn-Tg mice were produced using Imaris (Bitplane, d ). The number of p62 puncta or p62-positive/ h α-Syn-positive puncta was quantified in microglia at striatum of Atg7 flox/flox ; h α-Syn-Tg mice (n=5) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox ; h α-Syn-Tg mice (n=5) ( e ). p values were calculated by Mann–Whitney U test. Scale bar, 10µm. (f) After 7-months after tamoxifen, brains from Atg7 flox/flox ; h α-Syn-Tg mice (n=7) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 f lox/flox ; h α-Syn-Tg mice (n=6) were homogenized and fractionated into detergent-soluble fraction and detergent-insoluble fraction, and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP for confirming the presence of Cx3cr1 CreER-IRES-Eyfp . Band intensities were quantified in the lower panel. p values were calculated by Unpaired two-tailed Student’s t-test. (g-j) p-S129-α-synuclein-positive structures were visualized in brain sections from A tg7 flox/flox ; h α-Syn-Tg mice (n=7) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox ; h α-Syn-Tg mice (n=6) through DAB staining method ( g, i ), and quantified ( h, j ). p values were calculated by Unpaired two-tailed Student’s t-test. Scale bar, 100µm. All values are reported as mean ± SEM.

    Journal: bioRxiv

    Article Title: Microglia Clear Neuron-released α-Synuclein via Selective Autophagy and Prevent Neurodegeneration

    doi: 10.1101/2019.12.11.872812

    Figure Lengend Snippet: Disruption of autophagy in microglia promotes accumulation of misfolded α-synuclein species in mouse brain. (a) Experimental plan to test the roles of microglia-specific Atg7 -deficiency in AAV- h α-Syn mice (b) After 6-weeks after AAV- h α-Syn, brains from Atg7 flox/flox mice (n=4) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox mice (n=3) were homogenized and fractionated into a detergent-soluble fraction and detergent-insoluble fraction and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP. Actin were used as loading controls. Arrow indicates the EYFP band. (c) Experimental plan to test the roles of microglia-specific Atg7 -deficiency in h α-Syn-Tg mice (d, e) After 7-months after tamoxifen, brain slices were fixed and stained with antibodies against human α-synuclein, p62, and Iba-1. Representative 3D reconstruction pictures of microglia containing p62 and human α-synuclein in the striatum of Atg7 flox/flox mice and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox mice bred with h α-Syn-Tg mice were produced using Imaris (Bitplane, d ). The number of p62 puncta or p62-positive/ h α-Syn-positive puncta was quantified in microglia at striatum of Atg7 flox/flox ; h α-Syn-Tg mice (n=5) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox ; h α-Syn-Tg mice (n=5) ( e ). p values were calculated by Mann–Whitney U test. Scale bar, 10µm. (f) After 7-months after tamoxifen, brains from Atg7 flox/flox ; h α-Syn-Tg mice (n=7) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 f lox/flox ; h α-Syn-Tg mice (n=6) were homogenized and fractionated into detergent-soluble fraction and detergent-insoluble fraction, and assayed for W.B using antibodies against human α-synuclein, and GFP/YFP for confirming the presence of Cx3cr1 CreER-IRES-Eyfp . Band intensities were quantified in the lower panel. p values were calculated by Unpaired two-tailed Student’s t-test. (g-j) p-S129-α-synuclein-positive structures were visualized in brain sections from A tg7 flox/flox ; h α-Syn-Tg mice (n=7) and Cx3cr1 CreER-IRES-Eyfp ; Atg7 flox/flox ; h α-Syn-Tg mice (n=6) through DAB staining method ( g, i ), and quantified ( h, j ). p values were calculated by Unpaired two-tailed Student’s t-test. Scale bar, 100µm. All values are reported as mean ± SEM.

    Article Snippet: For cultured primary microglia, cells were fixed with 4% paraformaldehyde at 4□ for 15 minutes and incubated with microwave-boiled antigen retrieval solution (#CTS013, R & D System) for 5 min. After washing with PBS, cells were incubated with blocking/permeabilization buffer (1% BSA and 0.1%Triton X-100 in PBS) at RT for 30 minutes and incubated with primary antibodies including EEA1 (610457, BD Bioscience), α-synuclein (clone MJFR1, ab138501, Abcam; clone 2f12, MABN1817, Millipore; clone 42, 610787 BD Bioscience; clone 204, #2647, Cell Signaling), p62 (GP62-C, Progen, Germany), ubiquitin (clone P4D1, sc-8017, Santa Cruz Biotechnology), and TFEB (A303-673A, Bethyl Laboratories Inc, TX) at 4□ overnight followed by secondary antibodies.

    Techniques: Mouse Assay, Staining, Produced, MANN-WHITNEY, Two Tailed Test

    ROS produced by up-regulated MAOA plays an essential role for androgen deprivation-induced autophagy activation, NED and anti-apoptosis. ( A ) Immunoblotting of MAOA, LC3B-II and p62/SQSTM1 in shCtrl and shMAOA LNCaP cells treated as described in Fig. 3C for 48 and 96 hours. β-actin was used as loading control. Uncropped images are presented in Supplementary Figure S13A (right). ( B ) ROS production measured by OxiSelect In Vitro ROS/RNS Assay Kit in cells treated as described in ( A ). Data represent mean ± S.D. (n = 3); *** p

    Journal: Scientific Reports

    Article Title: MAOA-a novel decision maker of apoptosis and autophagy in hormone refractory neuroendocrine prostate cancer cells

    doi: 10.1038/srep46338

    Figure Lengend Snippet: ROS produced by up-regulated MAOA plays an essential role for androgen deprivation-induced autophagy activation, NED and anti-apoptosis. ( A ) Immunoblotting of MAOA, LC3B-II and p62/SQSTM1 in shCtrl and shMAOA LNCaP cells treated as described in Fig. 3C for 48 and 96 hours. β-actin was used as loading control. Uncropped images are presented in Supplementary Figure S13A (right). ( B ) ROS production measured by OxiSelect In Vitro ROS/RNS Assay Kit in cells treated as described in ( A ). Data represent mean ± S.D. (n = 3); *** p

    Article Snippet: 40 μg of TCLs was loaded onto 8–15% SDS-PAGE, transferred to the Hybond-C Extra membranes (Amersham Biosciences), and incubated with antibodies against proteins of interest, including β-tubulin III (1:5000, Cell Signaling Technology), LC3B (1:1000, Cell Signaling Technology), p62/SQSTM1 (1:1000, Cell Signaling Technology), full-length caspase 9 (1:1000, Cell Signaling Technology), cleaved caspase 9 (1:1000, Cell signaling technology), full-length caspase 3 (1:1000, Cell Signaling Technology), cleaved caspase 3 (1:1000, Cell signaling technology), phospho-p53 (Ser15) (1:1000, Cell Signaling Technology), p53 (1:1000, Cell Signaling Technology), REST (1:500, BD), MAOA (1:2000, Santa Cruz) and GAPDH (1:3000, Santa Cruz).

    Techniques: Produced, Activation Assay, In Vitro

    Morphine induces the degradation of PINK1 and the rescue of PINK1 improves the accumulation of SQSTM1/p62 induced by morphine. ( A ) SH-SY5Y cells were treated with CCCP (10 μM) for 2.5 h with or without morphine (200 μM) for 24 h. The level of PINK1 was analyzed by western blotting. n = 3, *** P

    Journal: Journal of Molecular Cell Biology

    Article Title: Morphine induces dysfunction of PINK1/Parkin-mediated mitophagy in spinal cord neurons implying involvement in antinociceptive tolerance

    doi: 10.1093/jmcb/mjz002

    Figure Lengend Snippet: Morphine induces the degradation of PINK1 and the rescue of PINK1 improves the accumulation of SQSTM1/p62 induced by morphine. ( A ) SH-SY5Y cells were treated with CCCP (10 μM) for 2.5 h with or without morphine (200 μM) for 24 h. The level of PINK1 was analyzed by western blotting. n = 3, *** P

    Article Snippet: Sections from each group were incubated with primary antibody overnight (NeuN, 1:300; IBA-1, 1:300; GFAP, 1:300; SQSTM1/p62, 1:100).

    Techniques: Western Blot

    The distribution and cellular location of SQSTM1/p62 after seven consecutive days of morphine intrathecal injection in the spinal cord dorsal horn. Sections of spinal cord from mice chronically administrated with morphine were fixed and subjected to double immunofluorescence stain. Confocal microscopy was performed to determine the co-localization of SQSTM1/p62 (green) and neuron (NeuN, red), microglia (IBA-1, red), or astrocyte (GFAP, red). Spinal samples were collected after the last administration of morphine. NeuN, neuronal nuclear protein; IBA-1, ionized calcium-binding adapter molecule 1; GFAP, glial fibrillary acidic protein. Scale bar, 100 μm.

    Journal: Journal of Molecular Cell Biology

    Article Title: Morphine induces dysfunction of PINK1/Parkin-mediated mitophagy in spinal cord neurons implying involvement in antinociceptive tolerance

    doi: 10.1093/jmcb/mjz002

    Figure Lengend Snippet: The distribution and cellular location of SQSTM1/p62 after seven consecutive days of morphine intrathecal injection in the spinal cord dorsal horn. Sections of spinal cord from mice chronically administrated with morphine were fixed and subjected to double immunofluorescence stain. Confocal microscopy was performed to determine the co-localization of SQSTM1/p62 (green) and neuron (NeuN, red), microglia (IBA-1, red), or astrocyte (GFAP, red). Spinal samples were collected after the last administration of morphine. NeuN, neuronal nuclear protein; IBA-1, ionized calcium-binding adapter molecule 1; GFAP, glial fibrillary acidic protein. Scale bar, 100 μm.

    Article Snippet: Sections from each group were incubated with primary antibody overnight (NeuN, 1:300; IBA-1, 1:300; GFAP, 1:300; SQSTM1/p62, 1:100).

    Techniques: Injection, Mouse Assay, Immunofluorescence, Staining, Confocal Microscopy, Binding Assay

    Morphine activates the initiation of autophagy and leads to the accumulation of SQSTM1/p62 protein. Repeated intrathecal morphine injections affected the protein levels of BECN1, Atg 5, LC3-II, and SQSTM1 in the spinal cord of mice. Representative western blots and densitometric analysis summary (normalized to ACTB/actin loading control) are shown. ( A ) Morphine did not change the level of BECN1 after 7 days of intrathecal injection. ( B and C ) Morphine increased the levels of Atg 5 and LC3-II after 1 day of intrathecal injection. ( D ) Morphine increased the level of SQSTM1/p62 after 5 days of intrathecal injection. n = 4, * P

    Journal: Journal of Molecular Cell Biology

    Article Title: Morphine induces dysfunction of PINK1/Parkin-mediated mitophagy in spinal cord neurons implying involvement in antinociceptive tolerance

    doi: 10.1093/jmcb/mjz002

    Figure Lengend Snippet: Morphine activates the initiation of autophagy and leads to the accumulation of SQSTM1/p62 protein. Repeated intrathecal morphine injections affected the protein levels of BECN1, Atg 5, LC3-II, and SQSTM1 in the spinal cord of mice. Representative western blots and densitometric analysis summary (normalized to ACTB/actin loading control) are shown. ( A ) Morphine did not change the level of BECN1 after 7 days of intrathecal injection. ( B and C ) Morphine increased the levels of Atg 5 and LC3-II after 1 day of intrathecal injection. ( D ) Morphine increased the level of SQSTM1/p62 after 5 days of intrathecal injection. n = 4, * P

    Article Snippet: Sections from each group were incubated with primary antibody overnight (NeuN, 1:300; IBA-1, 1:300; GFAP, 1:300; SQSTM1/p62, 1:100).

    Techniques: Mouse Assay, Western Blot, Injection

    Model for HSF1 and autophagy in cellular response to Hsp90 inhibitors. HSF1 is activated by Hsp90 inhibitors and enhances the activity of the transcription factor HSF1, which by promoting the expression of p62/SQSTM1 maintains autophagic and mitigates

    Journal: Biochemical pharmacology

    Article Title: Heat Shock Factor 1 Confers Resistance to Hsp90 Inhibitors through p62/SQSTM1 Expression and Promotion of Autophagic Flux

    doi: 10.1016/j.bcp.2013.11.014

    Figure Lengend Snippet: Model for HSF1 and autophagy in cellular response to Hsp90 inhibitors. HSF1 is activated by Hsp90 inhibitors and enhances the activity of the transcription factor HSF1, which by promoting the expression of p62/SQSTM1 maintains autophagic and mitigates

    Article Snippet: Thus, the HSF1-driven expression of p62/SQSTM1 contributes to mitigating the efficacy of Hsp90 inhibitors.

    Techniques: Activity Assay, Expressing

    HSF1-dependence of p62/SQSTM1 expression, and effect of silencing p62/SQSTM1 on cellular sensitivity to Hsp90 inhibitors. a. Real-time PCR data for p62/SQSTM1 mRNA expression in control (NEG siRNA) and HSF1-silenced (HSF1 siRNA) cells. Values for p62/SQSTM1

    Journal: Biochemical pharmacology

    Article Title: Heat Shock Factor 1 Confers Resistance to Hsp90 Inhibitors through p62/SQSTM1 Expression and Promotion of Autophagic Flux

    doi: 10.1016/j.bcp.2013.11.014

    Figure Lengend Snippet: HSF1-dependence of p62/SQSTM1 expression, and effect of silencing p62/SQSTM1 on cellular sensitivity to Hsp90 inhibitors. a. Real-time PCR data for p62/SQSTM1 mRNA expression in control (NEG siRNA) and HSF1-silenced (HSF1 siRNA) cells. Values for p62/SQSTM1

    Article Snippet: Thus, the HSF1-driven expression of p62/SQSTM1 contributes to mitigating the efficacy of Hsp90 inhibitors.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Momordicoside G selectively affects M1 macrophage phenotype and function in vitro . (A) Macrophage phenotypes analyzed by FITC-conjugated anti-mouse iNOS or arginase staining ( n = 10, 40×). (B) Cell viability examined by MTT ( n = 5). (C) ROS detected by DCFH-DA ( n = 5). (D) Cell autophagy-associated markers examined by Western blot ( n = 3). (E) Cell autophagy indicated by AO, LC3-B, and P62 staining ( n = 5, 40×). The data presents mean ± SD, the experiments were repeated 3 times, and statistical significance was determined by a t -test. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Momordicoside G Regulates Macrophage Phenotypes to Stimulate Efficient Repair of Lung Injury and Prevent Urethane-Induced Lung Carcinoma Lesions

    doi: 10.3389/fphar.2019.00321

    Figure Lengend Snippet: Momordicoside G selectively affects M1 macrophage phenotype and function in vitro . (A) Macrophage phenotypes analyzed by FITC-conjugated anti-mouse iNOS or arginase staining ( n = 10, 40×). (B) Cell viability examined by MTT ( n = 5). (C) ROS detected by DCFH-DA ( n = 5). (D) Cell autophagy-associated markers examined by Western blot ( n = 3). (E) Cell autophagy indicated by AO, LC3-B, and P62 staining ( n = 5, 40×). The data presents mean ± SD, the experiments were repeated 3 times, and statistical significance was determined by a t -test. ∗ P

    Article Snippet: Antibodies used herein including anti-NLRP3, anti-arginase, anti-CD68, anti-iNOS, anti- LC3-B, anti-p62, β-actin, and FITC-conjugated goat anti-mouse IgG were obtained from BD Pharmingen.

    Techniques: In Vitro, Staining, MTT Assay, Western Blot

    p62 and LC3 in freshly isolated human endothelial cells. A : Venous endothelial cells were isolated from healthy subjects and diabetic subjects as outlined in the methods. Endothelial cells from diabetic subjects (n=11) and non-diabetic controls (n=16)

    Journal: Atherosclerosis

    Article Title: Restoration of Autophagy in Endothelial Cells from Patients with Diabetes Mellitus Improves Nitric Oxide Signaling

    doi: 10.1016/j.atherosclerosis.2016.01.043

    Figure Lengend Snippet: p62 and LC3 in freshly isolated human endothelial cells. A : Venous endothelial cells were isolated from healthy subjects and diabetic subjects as outlined in the methods. Endothelial cells from diabetic subjects (n=11) and non-diabetic controls (n=16)

    Article Snippet: – Slides were stained with one of the following primary antibodies for one hour, unless otherwise indicated: p62 (1:100 dilution; Abnova, Cambridge, MA), beclin 1 (1:300 dilution; Novus Biologicals, Littleton, CO), LC3B (1:100; Abcam, Cambridge, MA), phosphorylated Ser1177 eNOS (1:200 dilution; Millipore), eNOS (1:100 dilution; BD Transduction Laboratories, San Diego, CA), Rab7 (1:100 dilution for 4 hours; Abcam), Lamp2a (1:100 dilution; Abcam), Atg7 (1:200 dilution; Sigma Aldrich, St. Louis, MO) and acetylated Lys9 histone 3 (1:100 dilution for 4 hours; Cell Signaling, Beverly, MA).

    Techniques: Isolation

    Autophagic flux is ongoing, but inadequate in endothelial cells from diabetic subjects. A : Bafilomycin increased p62 levels in endothelial cells from diabetic subjects assessed by immunofluorescence (n = 6, # P ≤0.01). B : Augmentation of p62 levels

    Journal: Atherosclerosis

    Article Title: Restoration of Autophagy in Endothelial Cells from Patients with Diabetes Mellitus Improves Nitric Oxide Signaling

    doi: 10.1016/j.atherosclerosis.2016.01.043

    Figure Lengend Snippet: Autophagic flux is ongoing, but inadequate in endothelial cells from diabetic subjects. A : Bafilomycin increased p62 levels in endothelial cells from diabetic subjects assessed by immunofluorescence (n = 6, # P ≤0.01). B : Augmentation of p62 levels

    Article Snippet: – Slides were stained with one of the following primary antibodies for one hour, unless otherwise indicated: p62 (1:100 dilution; Abnova, Cambridge, MA), beclin 1 (1:300 dilution; Novus Biologicals, Littleton, CO), LC3B (1:100; Abcam, Cambridge, MA), phosphorylated Ser1177 eNOS (1:200 dilution; Millipore), eNOS (1:100 dilution; BD Transduction Laboratories, San Diego, CA), Rab7 (1:100 dilution for 4 hours; Abcam), Lamp2a (1:100 dilution; Abcam), Atg7 (1:200 dilution; Sigma Aldrich, St. Louis, MO) and acetylated Lys9 histone 3 (1:100 dilution for 4 hours; Cell Signaling, Beverly, MA).

    Techniques: Immunofluorescence

    Autophagy markers in human aortic endothelial cells (HAEC’s) under diabetic conditions. A : p62 was increased in HAEC’s exposed to 30 mM glucose compared to cells exposed to 5 mM glucose (# P

    Journal: Atherosclerosis

    Article Title: Restoration of Autophagy in Endothelial Cells from Patients with Diabetes Mellitus Improves Nitric Oxide Signaling

    doi: 10.1016/j.atherosclerosis.2016.01.043

    Figure Lengend Snippet: Autophagy markers in human aortic endothelial cells (HAEC’s) under diabetic conditions. A : p62 was increased in HAEC’s exposed to 30 mM glucose compared to cells exposed to 5 mM glucose (# P

    Article Snippet: – Slides were stained with one of the following primary antibodies for one hour, unless otherwise indicated: p62 (1:100 dilution; Abnova, Cambridge, MA), beclin 1 (1:300 dilution; Novus Biologicals, Littleton, CO), LC3B (1:100; Abcam, Cambridge, MA), phosphorylated Ser1177 eNOS (1:200 dilution; Millipore), eNOS (1:100 dilution; BD Transduction Laboratories, San Diego, CA), Rab7 (1:100 dilution for 4 hours; Abcam), Lamp2a (1:100 dilution; Abcam), Atg7 (1:200 dilution; Sigma Aldrich, St. Louis, MO) and acetylated Lys9 histone 3 (1:100 dilution for 4 hours; Cell Signaling, Beverly, MA).

    Techniques:

    Exercise, AICAR and rapamycin counteract muscle wasting in cancer cachexia and modulate an adequate autophagic flux level. ( A ) In physiological conditions, a balance between autophagosome production and clearance maintains an adequate autophagic flux (red arrow) in the muscles, mirrored by basal levels of LC3bII (black dots) and p62 (green dots) expression. In cachectic muscles from C26-bearing mice ( B ), a strong accumulation of both LC3bII and p62 proteins points to an unbalanced autophagosome production/clearance ratio. This correlates with the pathophysiological features observed in cancer-related muscle wasting, including overexpression of Atrogin1 and Murf1 genes, as well as a decline in body weight (BD), muscle weight (MW), fiber size and muscle function. Spontaneous wheel running, or treatment with AICAR or rapamycin ( C ) counteracts cancer-related muscle wasting in tumor-bearing mice and induces a decrease in both LC3bII and p62 accumulation. Atrogin1 and Murf1 gene expression was restored to the basal levels observed in healthy muscles. This is associated with increased body and muscle weight and improved muscle function.

    Journal: Scientific Reports

    Article Title: Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer

    doi: 10.1038/srep26991

    Figure Lengend Snippet: Exercise, AICAR and rapamycin counteract muscle wasting in cancer cachexia and modulate an adequate autophagic flux level. ( A ) In physiological conditions, a balance between autophagosome production and clearance maintains an adequate autophagic flux (red arrow) in the muscles, mirrored by basal levels of LC3bII (black dots) and p62 (green dots) expression. In cachectic muscles from C26-bearing mice ( B ), a strong accumulation of both LC3bII and p62 proteins points to an unbalanced autophagosome production/clearance ratio. This correlates with the pathophysiological features observed in cancer-related muscle wasting, including overexpression of Atrogin1 and Murf1 genes, as well as a decline in body weight (BD), muscle weight (MW), fiber size and muscle function. Spontaneous wheel running, or treatment with AICAR or rapamycin ( C ) counteracts cancer-related muscle wasting in tumor-bearing mice and induces a decrease in both LC3bII and p62 accumulation. Atrogin1 and Murf1 gene expression was restored to the basal levels observed in healthy muscles. This is associated with increased body and muscle weight and improved muscle function.

    Article Snippet: The following primary antibody and dilutions were used: P-mTOR S2448 (Cell Signalling) 1:100; mTOR (Cell Signalling) 1:100; P-AMPKalpha T172 (Cell Signalling) 1: 1000; AMPKalpha (Cell Signalling) 1:1000; p-Acetyl-CoA Carbossylase S79 (Cell Signalling) 1:100; LC3b (Cell Signalling) 1:1000; p62 (Sigma) 1:10000; GAPDH (Santa Cruz) 1:10000; tubulin (Sigma) 1:10000.

    Techniques: Expressing, Mouse Assay, Over Expression

    Increased p62 expression of HCC and CCA patients carrying the rs146589465-G variant. Sections from 28 paraffin-embedded HCC or CCA patient samples were stained with a p62 antibody. Numbers correlate with patient numbering in Table S20 and an asterisk indicates an rs146589465-G carrier. Information on liver disease is indicated; Cirr, cirrhosis; H.C., hemochromatosis; HBV, Hepatitis B virus. Samples are grouped by International Classification of Diseases (ICD) diagnoses information from the Icelandic Cancer Registry.

    Journal: bioRxiv

    Article Title: Genetic risk of cholangiocarcinoma is linked to the autophagy gene ATG7

    doi: 10.1101/836767

    Figure Lengend Snippet: Increased p62 expression of HCC and CCA patients carrying the rs146589465-G variant. Sections from 28 paraffin-embedded HCC or CCA patient samples were stained with a p62 antibody. Numbers correlate with patient numbering in Table S20 and an asterisk indicates an rs146589465-G carrier. Information on liver disease is indicated; Cirr, cirrhosis; H.C., hemochromatosis; HBV, Hepatitis B virus. Samples are grouped by International Classification of Diseases (ICD) diagnoses information from the Icelandic Cancer Registry.

    Article Snippet: Tris/EDTA buffer pH 9 was used for the ATG7 (clone EP1759Y, Millipore 04-1055; 1:1000), LC3B (clone D11, Cell Signaling Technology 3868; 1:250) and p62 (Enzo PW9860; 1:100) antibodies in a 98.2°C water bath (5/20/20).

    Techniques: Expressing, Variant Assay, Staining

    The MG-132-mediated upregulation of SQSTM1/p62 protein expression requires the specific presence of ELAVL1/HuR protein. Representative western blotting (upper) and densitometric analysis (lower) of ELAVL1/HuR (A) and SQSTM1/p62 (B) proteins in the total homogenates of ELAVL1/HuR silenced ARPE-19 cells and negative control (NEG-siRNA) cells after exposure to 5 µM MG-132 for 24 h. α-tubulin was used as a loading control. Results are expressed as means ± S.E.M. *p

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: The MG-132-mediated upregulation of SQSTM1/p62 protein expression requires the specific presence of ELAVL1/HuR protein. Representative western blotting (upper) and densitometric analysis (lower) of ELAVL1/HuR (A) and SQSTM1/p62 (B) proteins in the total homogenates of ELAVL1/HuR silenced ARPE-19 cells and negative control (NEG-siRNA) cells after exposure to 5 µM MG-132 for 24 h. α-tubulin was used as a loading control. Results are expressed as means ± S.E.M. *p

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: Expressing, Western Blot, Negative Control

    Sections of foveomacular areas of age-matched control eyes for SQSTM1/p62, ubiquitin and ELAVL1/HuR. The extent of cytoplasmic immunopositivity for SQSTM1/p62, Bruch’s membrane immunopositivity for ubiquitin and the immunopositivity of nuclei of RPE cells for ELAVL1/HuR (foveomacular areas shown; A, B and C respectively) in the RPE cells (shown by arrows) was evaluated microscopically (no staining or positive staining). For SQSTM1/p62 there were only a few immunopositive cytoplasm’s randomly distributed through-out the RPE cell layer and the nuclei were negative. Bruch’s membrane (shown by arrowheads) was immunopositive for ubiquitin occasionally through-out the RPE cell layer in very small amounts while all of the nuclei were negative. Half of the nuclei showed minor immunopositivity for ELAVL1/HuR while the cytoplasm’s of RPE cells were negative. The foveomacular areas showed no difference in immunohistochemical stainings when compared to areas of perimacular and peripheral retina in all of the proteins studied. (Original magnifications of x 200 and in insets x 400).

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: Sections of foveomacular areas of age-matched control eyes for SQSTM1/p62, ubiquitin and ELAVL1/HuR. The extent of cytoplasmic immunopositivity for SQSTM1/p62, Bruch’s membrane immunopositivity for ubiquitin and the immunopositivity of nuclei of RPE cells for ELAVL1/HuR (foveomacular areas shown; A, B and C respectively) in the RPE cells (shown by arrows) was evaluated microscopically (no staining or positive staining). For SQSTM1/p62 there were only a few immunopositive cytoplasm’s randomly distributed through-out the RPE cell layer and the nuclei were negative. Bruch’s membrane (shown by arrowheads) was immunopositive for ubiquitin occasionally through-out the RPE cell layer in very small amounts while all of the nuclei were negative. Half of the nuclei showed minor immunopositivity for ELAVL1/HuR while the cytoplasm’s of RPE cells were negative. The foveomacular areas showed no difference in immunohistochemical stainings when compared to areas of perimacular and peripheral retina in all of the proteins studied. (Original magnifications of x 200 and in insets x 400).

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: Staining, Immunohistochemistry

    Sections of eyes with clinically diagnosed AMD immunostained for SQSTM1/p62. The extent of cytoplasmic immunopositivity in the retinal pigment epithelial cells (RPE, shown by arrows) and in the drusen was evaluated microscopically (no staining or positive staining) by selecting 5 mm long areas of foveomacular (A), perimacular (B) and peripheral (C) regions. The SQSTM1/p62 staining in the foveomacular areas was more extensive as compared to the perimacular and peripheral areas (B and C, respectively). The drusen (shown by asterisks) were mostly SQSTM1/p62 negative. The nuclei of RPE cells were SQSTM1/p62 negative. (Original magnifications of x 200 and in insets x 400; Bruch's membrane shown by arrow heads).

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: Sections of eyes with clinically diagnosed AMD immunostained for SQSTM1/p62. The extent of cytoplasmic immunopositivity in the retinal pigment epithelial cells (RPE, shown by arrows) and in the drusen was evaluated microscopically (no staining or positive staining) by selecting 5 mm long areas of foveomacular (A), perimacular (B) and peripheral (C) regions. The SQSTM1/p62 staining in the foveomacular areas was more extensive as compared to the perimacular and peripheral areas (B and C, respectively). The drusen (shown by asterisks) were mostly SQSTM1/p62 negative. The nuclei of RPE cells were SQSTM1/p62 negative. (Original magnifications of x 200 and in insets x 400; Bruch's membrane shown by arrow heads).

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: Staining

    SQSTM1/p62 protein, but not ELAVL1/HuR protein, is degraded by autophagy in ARPE-19 cells. Representative western blotting and densitometric analysis of SQSTM1/p62 (A) and ELAVL1/HuR (B) proteins in the total homogenates of ARPE-19 cells after exposure to bafilomycin (50 nM) or/and MG-132 (5 µM) for 24 h. α-tubulin was used as a loading control. Results are expressed as means ± S.D. The data were analyzed by ANOVA, followed by Mann-Whitney; **p

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: SQSTM1/p62 protein, but not ELAVL1/HuR protein, is degraded by autophagy in ARPE-19 cells. Representative western blotting and densitometric analysis of SQSTM1/p62 (A) and ELAVL1/HuR (B) proteins in the total homogenates of ARPE-19 cells after exposure to bafilomycin (50 nM) or/and MG-132 (5 µM) for 24 h. α-tubulin was used as a loading control. Results are expressed as means ± S.D. The data were analyzed by ANOVA, followed by Mann-Whitney; **p

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: Western Blot, MANN-WHITNEY

    SQSTM1/p62 transcript as a new target of the RNA-binding ELAVL1/HuR protein. (A): RNA-binding activity of ELAVL1/HuR evaluated by AlphaScreen technology. Saturation binding experiments investigated by titrating a series of biotinylated single-stranded (BITEG-) RNAs, including TNF neg and SQSTM1/p62 neg, that we designated as negative controls, against 1 nM of rELAVL1/HuR. Calculated dissociation constants (K d ) for TNFalpha (3.83±0.69 nM, R 2 = 0.97), and SQSTM1/p62 (30.85±13.22 nM R 2 = 0.91) are indicated. The plots represent mean ± SD of two independent experiments. (B): Saturation binding experiments as function of rELAVL1/HuR concentrations against four different type of RNA-substrates at 50 nM concentration. 1 nM of rELAVL1/HuR was enough to reach saturation of the binding. The plots represent Mean ± SD of two independent experiments. (C): Effect of MG-132 exposure on SQSTM1/p62 gene expression. Determination of SQSTM1/p62 mRNA by real-time qPCR in human ARPE-19 cells following treatments with solvent (control) or 5 µM MG-132 for 24 hrs. SQSTM1/p62 mRNA expression in control cells was taken as 100%. The values obtained from total cellular mRNA have been normalized to the level of RPL6 mRNA and expressed as mean ± S.E.M. ***p

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: SQSTM1/p62 transcript as a new target of the RNA-binding ELAVL1/HuR protein. (A): RNA-binding activity of ELAVL1/HuR evaluated by AlphaScreen technology. Saturation binding experiments investigated by titrating a series of biotinylated single-stranded (BITEG-) RNAs, including TNF neg and SQSTM1/p62 neg, that we designated as negative controls, against 1 nM of rELAVL1/HuR. Calculated dissociation constants (K d ) for TNFalpha (3.83±0.69 nM, R 2 = 0.97), and SQSTM1/p62 (30.85±13.22 nM R 2 = 0.91) are indicated. The plots represent mean ± SD of two independent experiments. (B): Saturation binding experiments as function of rELAVL1/HuR concentrations against four different type of RNA-substrates at 50 nM concentration. 1 nM of rELAVL1/HuR was enough to reach saturation of the binding. The plots represent Mean ± SD of two independent experiments. (C): Effect of MG-132 exposure on SQSTM1/p62 gene expression. Determination of SQSTM1/p62 mRNA by real-time qPCR in human ARPE-19 cells following treatments with solvent (control) or 5 µM MG-132 for 24 hrs. SQSTM1/p62 mRNA expression in control cells was taken as 100%. The values obtained from total cellular mRNA have been normalized to the level of RPL6 mRNA and expressed as mean ± S.E.M. ***p

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: RNA Binding Assay, Activity Assay, Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction

    MG-132-induced increase of SQSTM1/p62 but not ELAVL1/HuR protein levels is counteracted by AICAR treatment. Representative western blotting (upper) and densitometric analysis (lower) of SQSTM1/p62 (A), ELAVL1/HuR (B) and MAP1LC3A/LC3-II (C) proteins in the total homogenates of ARPE-19 cells after starvation or/and exposure to AICAR (2 mM) or/and MG-132 (5 µM) for 0,5 h, 3 h, 12 h and 24 h. α-tubulin was used as a loading control. Control cells were exposed only to solvent (DMSO). Results are expressed as means ± S.E.M. The data were analyzed by ANOVA, followed by Dunnett’s Multiple Comparison Test; *p

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: MG-132-induced increase of SQSTM1/p62 but not ELAVL1/HuR protein levels is counteracted by AICAR treatment. Representative western blotting (upper) and densitometric analysis (lower) of SQSTM1/p62 (A), ELAVL1/HuR (B) and MAP1LC3A/LC3-II (C) proteins in the total homogenates of ARPE-19 cells after starvation or/and exposure to AICAR (2 mM) or/and MG-132 (5 µM) for 0,5 h, 3 h, 12 h and 24 h. α-tubulin was used as a loading control. Control cells were exposed only to solvent (DMSO). Results are expressed as means ± S.E.M. The data were analyzed by ANOVA, followed by Dunnett’s Multiple Comparison Test; *p

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: Western Blot

    Colocalization of SQSTM1/p62 and MAP1LC3A/LC3 and position changes of SQSTM1/p62, revealed by confocal microscopy analysis. A light merge orange/yellow signal of colocalizing MAP1LC3A/LC3 (pDendra2-hLC3, green) and SQSTM1/p62 (pDsRed2-hp62, red) is detectable, usually near to the cell nuclei. Nuclei are stained with blue dye. The scale bar equals to 5 µm. (A): Confocal microscopy images of untreated control ARPE-19 cells and cells exposed to AICAR 2 mM or/and MG-132 5 µM for 24 h. A = Aggregates (B): Position of SQSTM1/p62 (pDendra2-hp62) in ARPE-19 cells, revealed by confocal microscopy analysis. SQSTM1/p62 in two different areas was photoconverted within approximately 7 sec (circled). Within 1 minute after photoconversion, the photoconverted SQSTM1/p62 is stationary. Cells have been exposed to 5 µM MG-132 for 24 h. N = Cell nucleus.

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: Colocalization of SQSTM1/p62 and MAP1LC3A/LC3 and position changes of SQSTM1/p62, revealed by confocal microscopy analysis. A light merge orange/yellow signal of colocalizing MAP1LC3A/LC3 (pDendra2-hLC3, green) and SQSTM1/p62 (pDsRed2-hp62, red) is detectable, usually near to the cell nuclei. Nuclei are stained with blue dye. The scale bar equals to 5 µm. (A): Confocal microscopy images of untreated control ARPE-19 cells and cells exposed to AICAR 2 mM or/and MG-132 5 µM for 24 h. A = Aggregates (B): Position of SQSTM1/p62 (pDendra2-hp62) in ARPE-19 cells, revealed by confocal microscopy analysis. SQSTM1/p62 in two different areas was photoconverted within approximately 7 sec (circled). Within 1 minute after photoconversion, the photoconverted SQSTM1/p62 is stationary. Cells have been exposed to 5 µM MG-132 for 24 h. N = Cell nucleus.

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: Confocal Microscopy, Staining, Size-exclusion Chromatography

    Summary of the results. The proteasome inhibitor MG-132 down-regulates (−) the ubiquitin-proteasome pathway (UPP). AMPK-activator AICAR and proteasome inhibitor MG-132 co-treatment activates (+) the autophagy. UPP inhibition significantly increases (+) SQSTM1/p62 protein levels, while autophagy induction during UPP inhibition robustly decreases (−) SQSTM1/p62 protein levels. Since SQSTM1/p62 localizes to aggregates, the decreasing of SQSTM1/p62 reveals its autophagy clearance. In addition, proteasome inhibition significantly increases ELAVL1/HuR protein levels by itself and during the autophagy induction (+). Proteasome inhibition induces a positive regulation of SQSTM1/p62 expression that occurs also at post-transcriptional level via ELAVL1/HuR protein (+). Activated autophagy is able to completely abolish the MG-132-induced protein aggregation (−), which, in turn improves the cell vitality. Ubiquitin is also found in intracellular aggregates in ARPE-19 cells as well as from drusens. In contrast, SQSTM1/p62 is found only in the intracellular aggregates in ARPE-19 cells, but not in drusens. However, SQSTM1/p62 levels were high in macular area of RPE cells revealing impaired autophagy. What is the relation between drusen and intracellular aggregates remains still unknown. Red lines with minus symbol represent inhibiting and decreasing events in ARPE-19 cells. Black lines and plus symbol represent activating and increasing events in the ARPE-19 cell. Zero (0) and dark blue line indicate neutral effects in the ARPE-19 cell. UPP: ubiquitin-proteasome pathway.

    Journal: PLoS ONE

    Article Title: Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

    doi: 10.1371/journal.pone.0069563

    Figure Lengend Snippet: Summary of the results. The proteasome inhibitor MG-132 down-regulates (−) the ubiquitin-proteasome pathway (UPP). AMPK-activator AICAR and proteasome inhibitor MG-132 co-treatment activates (+) the autophagy. UPP inhibition significantly increases (+) SQSTM1/p62 protein levels, while autophagy induction during UPP inhibition robustly decreases (−) SQSTM1/p62 protein levels. Since SQSTM1/p62 localizes to aggregates, the decreasing of SQSTM1/p62 reveals its autophagy clearance. In addition, proteasome inhibition significantly increases ELAVL1/HuR protein levels by itself and during the autophagy induction (+). Proteasome inhibition induces a positive regulation of SQSTM1/p62 expression that occurs also at post-transcriptional level via ELAVL1/HuR protein (+). Activated autophagy is able to completely abolish the MG-132-induced protein aggregation (−), which, in turn improves the cell vitality. Ubiquitin is also found in intracellular aggregates in ARPE-19 cells as well as from drusens. In contrast, SQSTM1/p62 is found only in the intracellular aggregates in ARPE-19 cells, but not in drusens. However, SQSTM1/p62 levels were high in macular area of RPE cells revealing impaired autophagy. What is the relation between drusen and intracellular aggregates remains still unknown. Red lines with minus symbol represent inhibiting and decreasing events in ARPE-19 cells. Black lines and plus symbol represent activating and increasing events in the ARPE-19 cell. Zero (0) and dark blue line indicate neutral effects in the ARPE-19 cell. UPP: ubiquitin-proteasome pathway.

    Article Snippet: Therefore, we wanted to evaluate whether binding of SQSTM1/p62 to aggregates is reversible or irreversible prior to autophagy.

    Techniques: Inhibition, Expressing

    Impaired autophagosome maturation leads to locomotion defects in 2-d-old adult flies. (A and B) Atg8a-positive autophagosomes and p62 aggregates accumulate in Syx17 mutant brains (B) compared with similarly aged controls (A). (C–E) No autophagosomes are found by EM in neurons of control adult flies (C). Large-scale accumulation of autophagosomes (arrowheads) is obvious in Syx17 mutant neurons (D). Autophagosome accumulation is rescued in Syx17 mutant neurons by transgenic expression of Syx17 (E). (F and G) Quantification of data presented in A and B (F) and C–E (G); n = 9 for A and B, and n = 4 for C–E. Error bars mark SDs. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila

    doi: 10.1083/jcb.201211160

    Figure Lengend Snippet: Impaired autophagosome maturation leads to locomotion defects in 2-d-old adult flies. (A and B) Atg8a-positive autophagosomes and p62 aggregates accumulate in Syx17 mutant brains (B) compared with similarly aged controls (A). (C–E) No autophagosomes are found by EM in neurons of control adult flies (C). Large-scale accumulation of autophagosomes (arrowheads) is obvious in Syx17 mutant neurons (D). Autophagosome accumulation is rescued in Syx17 mutant neurons by transgenic expression of Syx17 (E). (F and G) Quantification of data presented in A and B (F) and C–E (G); n = 9 for A and B, and n = 4 for C–E. Error bars mark SDs. ***, P

    Article Snippet: We used chicken anti-GFP (1:1,500; Invitrogen), rabbit anti-Atg5 (1:100; Sigma-Aldrich), rabbit anti-Atg8a (1:500; provided by K. Kohler, Eidgenössische Technische Hochschule Zürich, Zurich, Switzerland; ), rabbit anti-p62 (1:2,000; ), rabbit anti–active caspase 3 (1:300; Cell Signaling Technology), rat anti-Atg8a (1:300), and rat anti-Syx17 (1:300; this study) primary and Alexa Fluor 488 anti–chicken, Alexa Fluor 488 anti–rabbit, Alexa Fluor 568 anti–rat, and Alexa Fluor 647 anti–rabbit (all 1:1,500; Invitrogen) secondary antibodies.

    Techniques: Mutagenesis, Transgenic Assay, Expressing

    Autophagosomes accumulate upon loss of Syx17, usnp , or VAMP7 . (A–C) Increased numbers of Atg8a-positive autophagosomes are seen in Syx17 mutant (A), usnp (B), and VAMP7 (C) RNAi cells in starved larvae. (D–F) p62 aggregates accumulate in Syx17 mutant (D), usnp (E), and VAMP7 (F) RNAi cells in starved larvae. Note that Syx17 mutant cells are marked by lack of GFP expression in A and D, whereas RNAi cells express LAMP1-GFP in B, C, E, and F. (G) Western blots show increased autophagosome-associated, lipidated Atg8a-II levels in starved Syx17 and VAMP7 mutant larvae compared with controls. Accumulation of p62 in Syx17 and VAMP7 mutants is comparable to Atg7 mutants that are unable to lipidate Atg8a. Both the 34- and 40-kD isoforms of Syx17 disappear in Syx17[LL] mutants based on rat anti-Syx17 immunoblots, whereas faint bands are visible in Syx17[WH] mutant larvae. (H) Atg8a-II and p62 accumulate in well-fed Syx17 mutant adults compared with controls or Syx16 mutants (an additional control). Both Syx17-specific bands are missing from Syx17 mutant adults. (I–L) Numerous large autolysosomes (AL) and few double-membrane autophagosomes (arrowheads) are visible in ultrastructural images of control fat body cells from starved animals (I). Loss of Syx17 (J), usnp (K), or VAMP7 (L) function leads to accumulation of autophagosomes and lack of autolysosomes. (M–O) Quantification of data presented in A–C (M), D–F (N), and I–L (O). AP, autophagosome. n = 10 for A–F, and n = 4 for I–L. Error bars mark SDs. ***, P

    Journal: The Journal of Cell Biology

    Article Title: Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila

    doi: 10.1083/jcb.201211160

    Figure Lengend Snippet: Autophagosomes accumulate upon loss of Syx17, usnp , or VAMP7 . (A–C) Increased numbers of Atg8a-positive autophagosomes are seen in Syx17 mutant (A), usnp (B), and VAMP7 (C) RNAi cells in starved larvae. (D–F) p62 aggregates accumulate in Syx17 mutant (D), usnp (E), and VAMP7 (F) RNAi cells in starved larvae. Note that Syx17 mutant cells are marked by lack of GFP expression in A and D, whereas RNAi cells express LAMP1-GFP in B, C, E, and F. (G) Western blots show increased autophagosome-associated, lipidated Atg8a-II levels in starved Syx17 and VAMP7 mutant larvae compared with controls. Accumulation of p62 in Syx17 and VAMP7 mutants is comparable to Atg7 mutants that are unable to lipidate Atg8a. Both the 34- and 40-kD isoforms of Syx17 disappear in Syx17[LL] mutants based on rat anti-Syx17 immunoblots, whereas faint bands are visible in Syx17[WH] mutant larvae. (H) Atg8a-II and p62 accumulate in well-fed Syx17 mutant adults compared with controls or Syx16 mutants (an additional control). Both Syx17-specific bands are missing from Syx17 mutant adults. (I–L) Numerous large autolysosomes (AL) and few double-membrane autophagosomes (arrowheads) are visible in ultrastructural images of control fat body cells from starved animals (I). Loss of Syx17 (J), usnp (K), or VAMP7 (L) function leads to accumulation of autophagosomes and lack of autolysosomes. (M–O) Quantification of data presented in A–C (M), D–F (N), and I–L (O). AP, autophagosome. n = 10 for A–F, and n = 4 for I–L. Error bars mark SDs. ***, P

    Article Snippet: We used chicken anti-GFP (1:1,500; Invitrogen), rabbit anti-Atg5 (1:100; Sigma-Aldrich), rabbit anti-Atg8a (1:500; provided by K. Kohler, Eidgenössische Technische Hochschule Zürich, Zurich, Switzerland; ), rabbit anti-p62 (1:2,000; ), rabbit anti–active caspase 3 (1:300; Cell Signaling Technology), rat anti-Atg8a (1:300), and rat anti-Syx17 (1:300; this study) primary and Alexa Fluor 488 anti–chicken, Alexa Fluor 488 anti–rabbit, Alexa Fluor 568 anti–rat, and Alexa Fluor 647 anti–rabbit (all 1:1,500; Invitrogen) secondary antibodies.

    Techniques: Mutagenesis, Expressing, Western Blot

    Autophagy is suppressed in liver macrophages after chronic ethanol exposure. Liver macrophages were isolated from WT mice after 6 weeks of a paired liquid diet or a Lieber–DeCarli diet containing 5% ethanol (v/v). A , the experimental design for B and C. B , LC3B protein expression was determined by Western blotting. LC3BII levels were reduced in liver macrophages from ethanol-fed mice. C , isolated liver macrophages were treated with ammonium chloride and leupeptin ( AC/Leu ) for 2 h, and LC3B and p62 expression was examined by Western blotting. Similar results were obtained in four independent experiments. Representative results and their quantification are shown. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Murine macrophage autophagy protects against alcohol-induced liver injury by degrading interferon regulatory factor 1 (IRF1) and removing damaged mitochondria

    doi: 10.1074/jbc.RA119.007409

    Figure Lengend Snippet: Autophagy is suppressed in liver macrophages after chronic ethanol exposure. Liver macrophages were isolated from WT mice after 6 weeks of a paired liquid diet or a Lieber–DeCarli diet containing 5% ethanol (v/v). A , the experimental design for B and C. B , LC3B protein expression was determined by Western blotting. LC3BII levels were reduced in liver macrophages from ethanol-fed mice. C , isolated liver macrophages were treated with ammonium chloride and leupeptin ( AC/Leu ) for 2 h, and LC3B and p62 expression was examined by Western blotting. Similar results were obtained in four independent experiments. Representative results and their quantification are shown. *, p

    Article Snippet: The cells were then incubated overnight with primary antibodies, including anti-p62 (ProGen), and anti-IRF1 (Cell Signaling Technology).

    Techniques: Isolation, Mouse Assay, Expressing, Western Blot

    p62/SQSTM1 regulates cellular levels of IRF1 through autophagy in macrophages. A , BMDMs from WT-LC3B-GFP transgenic mice were treated with LPS (100 ng/ml) for 4 h. Immunofluorescence for IRF1 and p62 was performed. Cyan , green , red , and blue represent nucleus, LC3B, IRF1, and p62. White indicates co-localization of LC3B, IRF1, and p62. DAPI , 4′,6-diamidino-2-phenylindole. B–D , control and p62-silenced BMDMs were treated with LPS (100 ng/ml) for 4 h. B , nuclear ( Nuc ) and cytosolic ( Cyt ) fractions were separated. IRF1, HDAC1, and α-tubulin expression was determined by Western blotting. C and D , CCL5 ( C ) and CXCL10 ( D ) mRNA expression was determined by quantitative real-time PCR. Similar results were obtained in three independent experiments. Representative results are shown. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Murine macrophage autophagy protects against alcohol-induced liver injury by degrading interferon regulatory factor 1 (IRF1) and removing damaged mitochondria

    doi: 10.1074/jbc.RA119.007409

    Figure Lengend Snippet: p62/SQSTM1 regulates cellular levels of IRF1 through autophagy in macrophages. A , BMDMs from WT-LC3B-GFP transgenic mice were treated with LPS (100 ng/ml) for 4 h. Immunofluorescence for IRF1 and p62 was performed. Cyan , green , red , and blue represent nucleus, LC3B, IRF1, and p62. White indicates co-localization of LC3B, IRF1, and p62. DAPI , 4′,6-diamidino-2-phenylindole. B–D , control and p62-silenced BMDMs were treated with LPS (100 ng/ml) for 4 h. B , nuclear ( Nuc ) and cytosolic ( Cyt ) fractions were separated. IRF1, HDAC1, and α-tubulin expression was determined by Western blotting. C and D , CCL5 ( C ) and CXCL10 ( D ) mRNA expression was determined by quantitative real-time PCR. Similar results were obtained in three independent experiments. Representative results are shown. *, p

    Article Snippet: The cells were then incubated overnight with primary antibodies, including anti-p62 (ProGen), and anti-IRF1 (Cell Signaling Technology).

    Techniques: Transgenic Assay, Mouse Assay, Immunofluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Macrophage autophagy regulates cellular levels of IRF1. A–C , WT and Atg7 −/− (KO) BMDMs were treated with LPS (100 ng/ml) up to 10 h ( A ) and for 4 h ( B and C ). A , Atg7, IRF1, phospho-IRF-3, total IRF3, and p62 expression was determined by Western blotting. B , IRF3 and IRF1 mRNA expression was determined by quantitative real-time PCR. C , BMDMs from WT and Atg7 −/− (KO) LC3B-GFP transgenic mice were used for immunofluorescence. Green , red , and blue represent LC3B, IRF1, and nucleus, respectively. Yellow indicates co-localization of LC3B and IRF1. D , WT and IRF1 −/− BMDMs were treated with LPS (100 ng/ml) for 4 h. CCL5 and CXCL10 mRNA expression was determined by quantitative real-time PCR. E and F , WT, TRIF −/− , and MyD88 −/− BMDMs were treated with LPS (100 ng/ml) for 4 h. IRF1 mRNA and protein expression was determined by quantitative real-time PCR ( E ) and Western blotting ( F ), respectively. Similar results were obtained in three independent experiments. Representative results are shown. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Murine macrophage autophagy protects against alcohol-induced liver injury by degrading interferon regulatory factor 1 (IRF1) and removing damaged mitochondria

    doi: 10.1074/jbc.RA119.007409

    Figure Lengend Snippet: Macrophage autophagy regulates cellular levels of IRF1. A–C , WT and Atg7 −/− (KO) BMDMs were treated with LPS (100 ng/ml) up to 10 h ( A ) and for 4 h ( B and C ). A , Atg7, IRF1, phospho-IRF-3, total IRF3, and p62 expression was determined by Western blotting. B , IRF3 and IRF1 mRNA expression was determined by quantitative real-time PCR. C , BMDMs from WT and Atg7 −/− (KO) LC3B-GFP transgenic mice were used for immunofluorescence. Green , red , and blue represent LC3B, IRF1, and nucleus, respectively. Yellow indicates co-localization of LC3B and IRF1. D , WT and IRF1 −/− BMDMs were treated with LPS (100 ng/ml) for 4 h. CCL5 and CXCL10 mRNA expression was determined by quantitative real-time PCR. E and F , WT, TRIF −/− , and MyD88 −/− BMDMs were treated with LPS (100 ng/ml) for 4 h. IRF1 mRNA and protein expression was determined by quantitative real-time PCR ( E ) and Western blotting ( F ), respectively. Similar results were obtained in three independent experiments. Representative results are shown. *, p

    Article Snippet: The cells were then incubated overnight with primary antibodies, including anti-p62 (ProGen), and anti-IRF1 (Cell Signaling Technology).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transgenic Assay, Mouse Assay, Immunofluorescence

    Immunohistochemical staining and double-immunofluorescence labeled microscopic images of ECIs in RCCs. Immunohistochemical staining showed that the ECIs were immunopositive for P62 ( A ), NBR1 ( B ), LC3 ( C ), BECN1 ( D ), and ATG5 ( E ), and immunonegative for LCK (arrows in G ). Ub1 immunopositivity was detected in a small number of ECIs (arrows in F ). Double-immunofluorescence staining of ECIs demonstrated co-localization of P62 with NBR1, LC3, BECN1 or ATG5 in ECIs ( H ).

    Journal: Scientific Reports

    Article Title: Autophagy defects and related genetic variations in renal cell carcinoma with eosinophilic cytoplasmic inclusions

    doi: 10.1038/s41598-018-28369-y

    Figure Lengend Snippet: Immunohistochemical staining and double-immunofluorescence labeled microscopic images of ECIs in RCCs. Immunohistochemical staining showed that the ECIs were immunopositive for P62 ( A ), NBR1 ( B ), LC3 ( C ), BECN1 ( D ), and ATG5 ( E ), and immunonegative for LCK (arrows in G ). Ub1 immunopositivity was detected in a small number of ECIs (arrows in F ). Double-immunofluorescence staining of ECIs demonstrated co-localization of P62 with NBR1, LC3, BECN1 or ATG5 in ECIs ( H ).

    Article Snippet: Non-specific antibody-binding interactions were blocked in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (Sigma-Aldrich, A9647) and 0.1% Tween-20 (Sigma-Aldrich, 274348) for 1 h. Sections were then incubated with mixtures of two primary antibodies (P62 and NBR1, P62 and LC3, P62 and BECN1, P62 and ATG5, NBR1 and PEX14, NBR1 and CAT1, or NBR1 and GM130) at 4 °C overnight, at the following dilutions: mouse anti-P62 (1:150; Abcam, ab56416), rabbit anti-NBR1 (1:250; Proteintech, 16004-1-AP), mouse anti-NBR1 (1:40; Abcam, ab55474), rabbit anti-LC3 (1:25; Proteintech, 14600-1-AP), rabbit anti-BECN1 (1:25; Proteintech, 11306-1-AP), rabbit anti-ATG5 (1:30; Proteintech, 10181-2-AP), rabbit anti-CAT1 (1:50; Abcam, ab16731), rabbit anti-PEX14 (1:100; Proteintech, 10594-1-AP), and rabbit anti-GM130 (1:40; Proteintech, 11308-1-AP).

    Techniques: Immunohistochemistry, Staining, Immunofluorescence, Labeling, Electric Cell-substrate Impedance Sensing, Double Immunofluorescence Staining

    Three-dimensional reconstruction images of ECIs. ( A ) ECIs without NBR1 and peroxisomes. ( A – D ) ECIs comprising P62, BECN1, and NBR1 with peroxisomes partially or completely surrounding them.

    Journal: Scientific Reports

    Article Title: Autophagy defects and related genetic variations in renal cell carcinoma with eosinophilic cytoplasmic inclusions

    doi: 10.1038/s41598-018-28369-y

    Figure Lengend Snippet: Three-dimensional reconstruction images of ECIs. ( A ) ECIs without NBR1 and peroxisomes. ( A – D ) ECIs comprising P62, BECN1, and NBR1 with peroxisomes partially or completely surrounding them.

    Article Snippet: Non-specific antibody-binding interactions were blocked in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (Sigma-Aldrich, A9647) and 0.1% Tween-20 (Sigma-Aldrich, 274348) for 1 h. Sections were then incubated with mixtures of two primary antibodies (P62 and NBR1, P62 and LC3, P62 and BECN1, P62 and ATG5, NBR1 and PEX14, NBR1 and CAT1, or NBR1 and GM130) at 4 °C overnight, at the following dilutions: mouse anti-P62 (1:150; Abcam, ab56416), rabbit anti-NBR1 (1:250; Proteintech, 16004-1-AP), mouse anti-NBR1 (1:40; Abcam, ab55474), rabbit anti-LC3 (1:25; Proteintech, 14600-1-AP), rabbit anti-BECN1 (1:25; Proteintech, 11306-1-AP), rabbit anti-ATG5 (1:30; Proteintech, 10181-2-AP), rabbit anti-CAT1 (1:50; Abcam, ab16731), rabbit anti-PEX14 (1:100; Proteintech, 10594-1-AP), and rabbit anti-GM130 (1:40; Proteintech, 11308-1-AP).

    Techniques: Electric Cell-substrate Impedance Sensing

    ML-9 blocks autophagic flux. ( a ) Chloroquine does not increase LC3-II levels induced by elevated concentrations of ML-9. LNCaP cells were incubated in full, serum-starved or ML-9-containing media for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Densitometric quantitation showing autophagic flux (change in LC-II levels induced by chloroquine) is represented. Values represent means±S.E.M. n =3. ( b ) Chloroquine fails to increase the number of eGFP-positive puncta following ML-9 treatment. eGFP-LC3 expressing LNCaP cells were treated with full, serum-starved or 30 μ M ML-9-containing media for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Quantitation shown on the right represents means±S.D. GFP-positive puncta per cell ( n =50) from three independent experiments. ( c ) ML-9 induces p62 accumulation. LNCaP cells were treated with full, serum-starved or ML-9 containing media for 12 h. Densitometric quantitation for normalized p62 relative to Actin is shown. Values represent means±S.E.M. n =3. ( d ) ML-9 blocks starvation induced autophagic flux. LNCaP cells were treated with full, serum-starved, 30 μ M ML-9-containing full medium or 30 μ M ML-9-containing serum-starved medium for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Densitometric quantitation showing autophagic flux (change in LC-II levels induced by chloroquine) as well as normalized p62 relative to Actin is shown. Values represent means±S.E.M. n =3

    Journal: Cell Death & Disease

    Article Title: Identification of ML-9 as a lysosomotropic agent targeting autophagy and cell death

    doi: 10.1038/cddis.2014.156

    Figure Lengend Snippet: ML-9 blocks autophagic flux. ( a ) Chloroquine does not increase LC3-II levels induced by elevated concentrations of ML-9. LNCaP cells were incubated in full, serum-starved or ML-9-containing media for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Densitometric quantitation showing autophagic flux (change in LC-II levels induced by chloroquine) is represented. Values represent means±S.E.M. n =3. ( b ) Chloroquine fails to increase the number of eGFP-positive puncta following ML-9 treatment. eGFP-LC3 expressing LNCaP cells were treated with full, serum-starved or 30 μ M ML-9-containing media for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Quantitation shown on the right represents means±S.D. GFP-positive puncta per cell ( n =50) from three independent experiments. ( c ) ML-9 induces p62 accumulation. LNCaP cells were treated with full, serum-starved or ML-9 containing media for 12 h. Densitometric quantitation for normalized p62 relative to Actin is shown. Values represent means±S.E.M. n =3. ( d ) ML-9 blocks starvation induced autophagic flux. LNCaP cells were treated with full, serum-starved, 30 μ M ML-9-containing full medium or 30 μ M ML-9-containing serum-starved medium for 6 h in the absence or presence of 50 μ M chloroquine for the last hour. Densitometric quantitation showing autophagic flux (change in LC-II levels induced by chloroquine) as well as normalized p62 relative to Actin is shown. Values represent means±S.E.M. n =3

    Article Snippet: Guinea pig anti-p62 (GP62-C) was from PROGEN Biotechnik GmbH (Heidelberg, Germany).

    Techniques: Incubation, Quantitation Assay, Expressing