anti-lc3 Search Results


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  • 99
    Millipore anti lc3 antibody
    Gene expression profile and rescue of GOT1-null 143B cells with different metabolites. a Gene expression profile before glucose deprivation b , Gene expression profile 4 h after glucose deprivation. c Changes of <t>LC3-II</t> levels before and after glucose deprivation for 4 h. d Cell viability in different concentrations of glucose for 24 h. e Cell viability upon glutamine deprivation for 24 h. f Relative viabilities of wild type 143B and A549 cells in medium with glucose concentration at 4.5 g/L or 0 g/L in the presence of AOA at concentration of 5 mM. g Relative cell viability in medium supplemented with different metabolites (Glc: glucose; Gly: glycine; Ser: serine; Gal: galactose). h Illustration of intermediates in gluconeogenesis pathway. i Wild type and GOT1-null 143B cells grown in glucose free medium supplemented with 10 mM aspartate (Asp), 5 mM OAA, or 2.5 mM PEP for 4 h, j , 16 h and k, 24 h. All the experiments have been repeated three times, and data are represented as mean ± s.d. One-way ANOVA test was performed for d , g , i , j and k . Unpaired student’s t-test was performed for a , b , e and f . *** p
    Anti Lc3 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti lc3
    Down-regulation of OGT promotes the formation of autolysosome induced by cisplatin. (A) OGT-deficient A2780 and SKOV3 cells were cultured in different concentrations of cisplatin with 3-MA (3 mM) or Baf (200 nM) for 48 h. Cell viability was measured by CCK-8. (B) OGT-deficient A2780 and SKOV3 cells were treated with 3-MA (3 mM) or Baf (200 nM) in the presence of cisplatin (5 µg/mL) for 24 h. Apoptotic cells were identified by ANXA5 and PI staining. (C-D) Control and OGT-deficient A2780 (C) and SKOV3 (D) cells were transfected with <t>mRFP-GFP-LC3</t> vector for 24 h and then treated with cisplatin (5 µg/mL) for 24 h. Representative images of fluorescent LC3 puncta are shown. The mean number of yellow puncta representing autophagosomes and the mean number of red puncta representing autolysosomes are plotted. * P
    Anti Lc3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti lc3b antibody
    Down-regulation of OGT promotes the formation of autolysosome induced by cisplatin. (A) OGT-deficient A2780 and SKOV3 cells were cultured in different concentrations of cisplatin with 3-MA (3 mM) or Baf (200 nM) for 48 h. Cell viability was measured by CCK-8. (B) OGT-deficient A2780 and SKOV3 cells were treated with 3-MA (3 mM) or Baf (200 nM) in the presence of cisplatin (5 µg/mL) for 24 h. Apoptotic cells were identified by ANXA5 and PI staining. (C-D) Control and OGT-deficient A2780 (C) and SKOV3 (D) cells were transfected with <t>mRFP-GFP-LC3</t> vector for 24 h and then treated with cisplatin (5 µg/mL) for 24 h. Representative images of fluorescent LC3 puncta are shown. The mean number of yellow puncta representing autophagosomes and the mean number of red puncta representing autolysosomes are plotted. * P
    Anti Lc3b Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Novus Biologicals lc3b antibody
    Down-regulation of OGT promotes the formation of autolysosome induced by cisplatin. (A) OGT-deficient A2780 and SKOV3 cells were cultured in different concentrations of cisplatin with 3-MA (3 mM) or Baf (200 nM) for 48 h. Cell viability was measured by CCK-8. (B) OGT-deficient A2780 and SKOV3 cells were treated with 3-MA (3 mM) or Baf (200 nM) in the presence of cisplatin (5 µg/mL) for 24 h. Apoptotic cells were identified by ANXA5 and PI staining. (C-D) Control and OGT-deficient A2780 (C) and SKOV3 (D) cells were transfected with <t>mRFP-GFP-LC3</t> vector for 24 h and then treated with cisplatin (5 µg/mL) for 24 h. Representative images of fluorescent LC3 puncta are shown. The mean number of yellow puncta representing autophagosomes and the mean number of red puncta representing autolysosomes are plotted. * P
    Lc3b Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 98/100, based on 1583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti lc3
    MHCD ameliorates Doxorubicin-induced increases in autophagy by inhibiting <t>LC3</t> and beclin-1 protein levels. (A) The expression of LC3 and beclin-1 in the glomeruli of Doxorubicin-induced nephrotic rats was assessed using immunohistochemistry (magnification, ×400). Quantification of (B) beclin-1 and (C) LC3. Data are expressed the mean ± standard deviation (n=10). *P
    Anti Lc3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Novus Biologicals anti lc3
    TNP-1 induced autophagy in a shape- and composition-dependent manner. a Fluorescent microscopy images of <t>EGFP-LC3/HeLa</t> cells treated with PBS (control) or 10 µg mL −1 of CuPd nanoparticles for 24 h. Scale bar, 10 µm. The right panel shows the quantified results for the percentage of cells containing at least 5 EGFP-LC3 dots. Mean ± s.e.m. n = 5. *** p
    Anti Lc3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 425 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    MBL International anti lc3
    Schematic model depicting the IFN-γ–mediated LAP-like process against liver-stage P. vivax . P. vivax sporozoites actively invade human hepatocytes and then reside within the parasitophorous vacuoles. IFN-γ treatment leads to the decoration of the parasitophorous vacuole membrane (PVM) with host <t>LC3,</t> in a process involving Beclin 1, PI3K, and Atg5. The LC3-decorated PVMs then fuse with lysosomes, which kills the parasite.
    Anti Lc3, supplied by MBL International, used in various techniques. Bioz Stars score: 93/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti lc3
    <t>LC3</t> activation is required for melanogenesis. Inhibition of LC3 attenuates melanin synthesis and tyrosinase activity in melanocytes. Melan-a cells were transfected with specific siRNAs for LC3, beclin-1, ATG5, or control siRNA for 24 h and were analyzed by Western blotting after autophagy induction (a,b) . Quantification of melanin (c) and tyrosinase activity (d) was performed for each transfected cell line. Results shown are the mean of three independent experiments ± SD. * P
    Anti Lc3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    WuXi AppTec anti lc3
    In vivo accumulation of <t>LC3</t> and LAMP-1 around T. gondii in brain endothelial cells and effects of administration of an inhibitor of autophagy on hematogenous invasion of the brain and eye by T. gondii . ( a ) Mice received T. gondii -infected dendritic cells i.v. After 18 h, mice were perfused and brain sections were stained with Tomato lectin-DyLight 488, anti- T. gondii Ab plus Alexa 568-conjugated secondary Ab and anti-LC3 or anti-LAMP-1 Abs plus Alexa 647-conjugated secondary Abs. Images to the left show parasites present in Tomato lectin + cells (endothelial cells). The images from the Trg-Ctr mouse show a parasite not surrounded by accumulation of LC3 or LAMP-1. Ring-like accumulation of LC3 or LAMP-1 around parasite (arrowheads) is noted in images from the Trg-DN EGFR mouse (X630). Bar, 5 μm. Bar graphs represent percentages of parasites present in endothelial cells that were surrounded by accumulation of LC3 or LAMP-1 (mean ± SEM from 2 pooled experiments). ** p
    Anti Lc3, supplied by WuXi AppTec, used in various techniques. Bioz Stars score: 97/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Novus Biologicals lc3a antibody
    In vivo accumulation of <t>LC3</t> and LAMP-1 around T. gondii in brain endothelial cells and effects of administration of an inhibitor of autophagy on hematogenous invasion of the brain and eye by T. gondii . ( a ) Mice received T. gondii -infected dendritic cells i.v. After 18 h, mice were perfused and brain sections were stained with Tomato lectin-DyLight 488, anti- T. gondii Ab plus Alexa 568-conjugated secondary Ab and anti-LC3 or anti-LAMP-1 Abs plus Alexa 647-conjugated secondary Abs. Images to the left show parasites present in Tomato lectin + cells (endothelial cells). The images from the Trg-Ctr mouse show a parasite not surrounded by accumulation of LC3 or LAMP-1. Ring-like accumulation of LC3 or LAMP-1 around parasite (arrowheads) is noted in images from the Trg-DN EGFR mouse (X630). Bar, 5 μm. Bar graphs represent percentages of parasites present in endothelial cells that were surrounded by accumulation of LC3 or LAMP-1 (mean ± SEM from 2 pooled experiments). ** p
    Lc3a Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit polyclonal anti lc3
    In vivo accumulation of <t>LC3</t> and LAMP-1 around T. gondii in brain endothelial cells and effects of administration of an inhibitor of autophagy on hematogenous invasion of the brain and eye by T. gondii . ( a ) Mice received T. gondii -infected dendritic cells i.v. After 18 h, mice were perfused and brain sections were stained with Tomato lectin-DyLight 488, anti- T. gondii Ab plus Alexa 568-conjugated secondary Ab and anti-LC3 or anti-LAMP-1 Abs plus Alexa 647-conjugated secondary Abs. Images to the left show parasites present in Tomato lectin + cells (endothelial cells). The images from the Trg-Ctr mouse show a parasite not surrounded by accumulation of LC3 or LAMP-1. Ring-like accumulation of LC3 or LAMP-1 around parasite (arrowheads) is noted in images from the Trg-DN EGFR mouse (X630). Bar, 5 μm. Bar graphs represent percentages of parasites present in endothelial cells that were surrounded by accumulation of LC3 or LAMP-1 (mean ± SEM from 2 pooled experiments). ** p
    Rabbit Polyclonal Anti Lc3, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti lc3 ii
    In vivo accumulation of <t>LC3</t> and LAMP-1 around T. gondii in brain endothelial cells and effects of administration of an inhibitor of autophagy on hematogenous invasion of the brain and eye by T. gondii . ( a ) Mice received T. gondii -infected dendritic cells i.v. After 18 h, mice were perfused and brain sections were stained with Tomato lectin-DyLight 488, anti- T. gondii Ab plus Alexa 568-conjugated secondary Ab and anti-LC3 or anti-LAMP-1 Abs plus Alexa 647-conjugated secondary Abs. Images to the left show parasites present in Tomato lectin + cells (endothelial cells). The images from the Trg-Ctr mouse show a parasite not surrounded by accumulation of LC3 or LAMP-1. Ring-like accumulation of LC3 or LAMP-1 around parasite (arrowheads) is noted in images from the Trg-DN EGFR mouse (X630). Bar, 5 μm. Bar graphs represent percentages of parasites present in endothelial cells that were surrounded by accumulation of LC3 or LAMP-1 (mean ± SEM from 2 pooled experiments). ** p
    Anti Lc3 Ii, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit polyclonal anti lc3
    In vivo accumulation of <t>LC3</t> and LAMP-1 around T. gondii in brain endothelial cells and effects of administration of an inhibitor of autophagy on hematogenous invasion of the brain and eye by T. gondii . ( a ) Mice received T. gondii -infected dendritic cells i.v. After 18 h, mice were perfused and brain sections were stained with Tomato lectin-DyLight 488, anti- T. gondii Ab plus Alexa 568-conjugated secondary Ab and anti-LC3 or anti-LAMP-1 Abs plus Alexa 647-conjugated secondary Abs. Images to the left show parasites present in Tomato lectin + cells (endothelial cells). The images from the Trg-Ctr mouse show a parasite not surrounded by accumulation of LC3 or LAMP-1. Ring-like accumulation of LC3 or LAMP-1 around parasite (arrowheads) is noted in images from the Trg-DN EGFR mouse (X630). Bar, 5 μm. Bar graphs represent percentages of parasites present in endothelial cells that were surrounded by accumulation of LC3 or LAMP-1 (mean ± SEM from 2 pooled experiments). ** p
    Rabbit Polyclonal Anti Lc3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Gene expression profile and rescue of GOT1-null 143B cells with different metabolites. a Gene expression profile before glucose deprivation b , Gene expression profile 4 h after glucose deprivation. c Changes of LC3-II levels before and after glucose deprivation for 4 h. d Cell viability in different concentrations of glucose for 24 h. e Cell viability upon glutamine deprivation for 24 h. f Relative viabilities of wild type 143B and A549 cells in medium with glucose concentration at 4.5 g/L or 0 g/L in the presence of AOA at concentration of 5 mM. g Relative cell viability in medium supplemented with different metabolites (Glc: glucose; Gly: glycine; Ser: serine; Gal: galactose). h Illustration of intermediates in gluconeogenesis pathway. i Wild type and GOT1-null 143B cells grown in glucose free medium supplemented with 10 mM aspartate (Asp), 5 mM OAA, or 2.5 mM PEP for 4 h, j , 16 h and k, 24 h. All the experiments have been repeated three times, and data are represented as mean ± s.d. One-way ANOVA test was performed for d , g , i , j and k . Unpaired student’s t-test was performed for a , b , e and f . *** p

    Journal: BMC Cancer

    Article Title: Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose

    doi: 10.1186/s12885-018-4443-1

    Figure Lengend Snippet: Gene expression profile and rescue of GOT1-null 143B cells with different metabolites. a Gene expression profile before glucose deprivation b , Gene expression profile 4 h after glucose deprivation. c Changes of LC3-II levels before and after glucose deprivation for 4 h. d Cell viability in different concentrations of glucose for 24 h. e Cell viability upon glutamine deprivation for 24 h. f Relative viabilities of wild type 143B and A549 cells in medium with glucose concentration at 4.5 g/L or 0 g/L in the presence of AOA at concentration of 5 mM. g Relative cell viability in medium supplemented with different metabolites (Glc: glucose; Gly: glycine; Ser: serine; Gal: galactose). h Illustration of intermediates in gluconeogenesis pathway. i Wild type and GOT1-null 143B cells grown in glucose free medium supplemented with 10 mM aspartate (Asp), 5 mM OAA, or 2.5 mM PEP for 4 h, j , 16 h and k, 24 h. All the experiments have been repeated three times, and data are represented as mean ± s.d. One-way ANOVA test was performed for d , g , i , j and k . Unpaired student’s t-test was performed for a , b , e and f . *** p

    Article Snippet: To study autophagy in GOT1-null cells, the primary antibody was anti-LC3 (Sigma, L8918), and the secondary antibody was donkey anti-rabbit (Santa Cruz).

    Techniques: Expressing, Concentration Assay, Gas Chromatography

    LC3-I/II as well as p62 protein expression levels ( A ) upon autophagy induction, using rapamycin or spermidine, as well as partial inhibition, using non-saturating concentrations of bafilomycin. A representative blot is shown. A minimum of three independent experiments ( n = 3) were performed. Autophagy pathway intermediate pool size quantification ( B ) and representative micrographs ( C ) of non-patterned and patterned cells.

    Journal: Cells

    Article Title: The Precision Control of Autophagic Flux and Vesicle Dynamics—A Micropattern Approach

    doi: 10.3390/cells7080094

    Figure Lengend Snippet: LC3-I/II as well as p62 protein expression levels ( A ) upon autophagy induction, using rapamycin or spermidine, as well as partial inhibition, using non-saturating concentrations of bafilomycin. A representative blot is shown. A minimum of three independent experiments ( n = 3) were performed. Autophagy pathway intermediate pool size quantification ( B ) and representative micrographs ( C ) of non-patterned and patterned cells.

    Article Snippet: The primary antibodies used were rabbit anti-LC3 (Sigma, L-8918 and anti-Sequestosome1/p62 (Abcam, Pretoria, South Africa, ab-56416).

    Techniques: Expressing, Inhibition

    Testicular regulation of autophagic and apoptotic-related genes in nutritional stress conditions. (A) Adult active testes were incubated in rich or starvation media and the mRNA expression of BECN1 and LC3 levels were measured. (B) Expression of testicular AI in adult active testes incubated in reach or starvation media. Values are expressed as mean ± SEM. * indicates significant differences between media for each incubation time ( p

    Journal: PLoS ONE

    Article Title: The balance between apoptosis and autophagy regulates testis regression and recrudescence in the seasonal-breeding South American plains vizcacha, Lagostomus maximus

    doi: 10.1371/journal.pone.0191126

    Figure Lengend Snippet: Testicular regulation of autophagic and apoptotic-related genes in nutritional stress conditions. (A) Adult active testes were incubated in rich or starvation media and the mRNA expression of BECN1 and LC3 levels were measured. (B) Expression of testicular AI in adult active testes incubated in reach or starvation media. Values are expressed as mean ± SEM. * indicates significant differences between media for each incubation time ( p

    Article Snippet: Membranes were blocked for 1 h in PBS with 5% non-fat dry milk and incubated with rabbit anti-LC3 antibody (1:1000, Sigma, Saint Louis, Missouri, USA).

    Techniques: Incubation, Expressing

    Localization and expression of autophagic- related genes/proteins in the testis of L . maximus during the annual reproductive cycle. Immunostaning (A) and mRNA expression (B) for BECN1 and LC3 and (C) LC3I/II protein levels in adult active, inactivating, inactive and activating testis. ST: seminiferous tubules, SG: spermatogonia; PS: primary spermatocyte; SS: secondary spermatocyte; SP: spermatid; SPZ: spermatozoid; SC: Sertoli cell. Scale bar: 50 μm. Values indicate mean ± SEM. Different letters indicate significant differences between groups ( p

    Journal: PLoS ONE

    Article Title: The balance between apoptosis and autophagy regulates testis regression and recrudescence in the seasonal-breeding South American plains vizcacha, Lagostomus maximus

    doi: 10.1371/journal.pone.0191126

    Figure Lengend Snippet: Localization and expression of autophagic- related genes/proteins in the testis of L . maximus during the annual reproductive cycle. Immunostaning (A) and mRNA expression (B) for BECN1 and LC3 and (C) LC3I/II protein levels in adult active, inactivating, inactive and activating testis. ST: seminiferous tubules, SG: spermatogonia; PS: primary spermatocyte; SS: secondary spermatocyte; SP: spermatid; SPZ: spermatozoid; SC: Sertoli cell. Scale bar: 50 μm. Values indicate mean ± SEM. Different letters indicate significant differences between groups ( p

    Article Snippet: Membranes were blocked for 1 h in PBS with 5% non-fat dry milk and incubated with rabbit anti-LC3 antibody (1:1000, Sigma, Saint Louis, Missouri, USA).

    Techniques: Expressing

    Down-regulation of OGT promotes the formation of autolysosome induced by cisplatin. (A) OGT-deficient A2780 and SKOV3 cells were cultured in different concentrations of cisplatin with 3-MA (3 mM) or Baf (200 nM) for 48 h. Cell viability was measured by CCK-8. (B) OGT-deficient A2780 and SKOV3 cells were treated with 3-MA (3 mM) or Baf (200 nM) in the presence of cisplatin (5 µg/mL) for 24 h. Apoptotic cells were identified by ANXA5 and PI staining. (C-D) Control and OGT-deficient A2780 (C) and SKOV3 (D) cells were transfected with mRFP-GFP-LC3 vector for 24 h and then treated with cisplatin (5 µg/mL) for 24 h. Representative images of fluorescent LC3 puncta are shown. The mean number of yellow puncta representing autophagosomes and the mean number of red puncta representing autolysosomes are plotted. * P

    Journal: Theranostics

    Article Title: Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer

    doi: 10.7150/thno.27806

    Figure Lengend Snippet: Down-regulation of OGT promotes the formation of autolysosome induced by cisplatin. (A) OGT-deficient A2780 and SKOV3 cells were cultured in different concentrations of cisplatin with 3-MA (3 mM) or Baf (200 nM) for 48 h. Cell viability was measured by CCK-8. (B) OGT-deficient A2780 and SKOV3 cells were treated with 3-MA (3 mM) or Baf (200 nM) in the presence of cisplatin (5 µg/mL) for 24 h. Apoptotic cells were identified by ANXA5 and PI staining. (C-D) Control and OGT-deficient A2780 (C) and SKOV3 (D) cells were transfected with mRFP-GFP-LC3 vector for 24 h and then treated with cisplatin (5 µg/mL) for 24 h. Representative images of fluorescent LC3 puncta are shown. The mean number of yellow puncta representing autophagosomes and the mean number of red puncta representing autolysosomes are plotted. * P

    Article Snippet: Cells were blocked with 0.1% BSA for 1 h at room temperature and subsequently incubated with anti-LC3 (1:100, CST, 2775s) and Lamp1 (1:200, Abcam, ab25630) at 4 °C overnight.

    Techniques: Cell Culture, CCK-8 Assay, Staining, Transfection, Plasmid Preparation

    O -GlcNAcylation of SNAP-29 regulates the interaction of SNAP-29 with Stx17 and VAMP8. (A) SKOV3 cells were transfected with SNAP-29 and SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The O -GlcNAcylation levels of SNAP-29 were tested by co-immunoprecipitation. (B) SKOV3 cells were transfected with SNAP-29-GFP, SNAP-29 (Mut)-GFP and RFP-LC3 vectors and then treated with cisplatin (5 µg/mL) for 24 h. Colocalization of SNAP-29 and LC3 was determined by confocal fluorescence microscopy. Scale bar represent 10 μm. (C) SKOV3 cells were transfected with control, SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The expressions of LC3 and p62 were measured by western blotting. (D) The expressions of LC3 and p62 in SKOV3 cells that were transfected with si Stx17 or si VAMP8 and treated with cisplatin (5 µg/mL) for 24 h. (E) SKOV3 cell were transfected with SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. Then, cell extracts were immunoprecipitated with anti-SNAP-29 and the resulting precipitants were immunoblotted against Stx17 and VAMP8. Whole-cell lysates were tested for SNAP-29 and actin. The values are presented as mean ± SD (n = 3). ** P

    Journal: Theranostics

    Article Title: Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer

    doi: 10.7150/thno.27806

    Figure Lengend Snippet: O -GlcNAcylation of SNAP-29 regulates the interaction of SNAP-29 with Stx17 and VAMP8. (A) SKOV3 cells were transfected with SNAP-29 and SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The O -GlcNAcylation levels of SNAP-29 were tested by co-immunoprecipitation. (B) SKOV3 cells were transfected with SNAP-29-GFP, SNAP-29 (Mut)-GFP and RFP-LC3 vectors and then treated with cisplatin (5 µg/mL) for 24 h. Colocalization of SNAP-29 and LC3 was determined by confocal fluorescence microscopy. Scale bar represent 10 μm. (C) SKOV3 cells were transfected with control, SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. The expressions of LC3 and p62 were measured by western blotting. (D) The expressions of LC3 and p62 in SKOV3 cells that were transfected with si Stx17 or si VAMP8 and treated with cisplatin (5 µg/mL) for 24 h. (E) SKOV3 cell were transfected with SNAP-29 or SNAP-29 (Mut) vectors and then treated with cisplatin (5 µg/mL) for 24 h. Then, cell extracts were immunoprecipitated with anti-SNAP-29 and the resulting precipitants were immunoblotted against Stx17 and VAMP8. Whole-cell lysates were tested for SNAP-29 and actin. The values are presented as mean ± SD (n = 3). ** P

    Article Snippet: Cells were blocked with 0.1% BSA for 1 h at room temperature and subsequently incubated with anti-LC3 (1:100, CST, 2775s) and Lamp1 (1:200, Abcam, ab25630) at 4 °C overnight.

    Techniques: Transfection, Immunoprecipitation, Fluorescence, Microscopy, Western Blot

    Down-regulation of OGT enhances autophagy induced by cisplatin in ovarian cancer cells. (A-B) A2780 and SKOV3 cells were treated with or without cisplatin (5 µg/mL) for 24 h. The expression levels of LC3 and p62 were tested by western blotting. (C-D) Control and OGT-deficient ovarian cancer cells were treated with cisplatin (5 µg/mL) for 24 h. LC3 and p62 expression levels were determined by western blotting. (E) Control and OGT-deficient ovarian cancer cells were cultured with cisplatin (5 µg/mL) for 24 h. LC3 puncta were detected by anti-LC3 by fluorescence microscopy. Scale bar represent 50 μm. The values are presented as mean ± SD (n = 3). ** P

    Journal: Theranostics

    Article Title: Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer

    doi: 10.7150/thno.27806

    Figure Lengend Snippet: Down-regulation of OGT enhances autophagy induced by cisplatin in ovarian cancer cells. (A-B) A2780 and SKOV3 cells were treated with or without cisplatin (5 µg/mL) for 24 h. The expression levels of LC3 and p62 were tested by western blotting. (C-D) Control and OGT-deficient ovarian cancer cells were treated with cisplatin (5 µg/mL) for 24 h. LC3 and p62 expression levels were determined by western blotting. (E) Control and OGT-deficient ovarian cancer cells were cultured with cisplatin (5 µg/mL) for 24 h. LC3 puncta were detected by anti-LC3 by fluorescence microscopy. Scale bar represent 50 μm. The values are presented as mean ± SD (n = 3). ** P

    Article Snippet: Cells were blocked with 0.1% BSA for 1 h at room temperature and subsequently incubated with anti-LC3 (1:100, CST, 2775s) and Lamp1 (1:200, Abcam, ab25630) at 4 °C overnight.

    Techniques: Expressing, Western Blot, Cell Culture, Fluorescence, Microscopy

    The O -GlcNAcylation level of SNAP-29 is associated with autophagy activity induced by cisplatin. (A) Control and OGT-deficient SKOV3 cells were transfected with NC siRNA or SNAP-29 siRNA and then treated with cisplatin (5 µg/mL) for 24 h. The expression levels of LC3 and p62 were examined by western blotting. (B) OGT-deficient SKOV3 cells were transfected with mRFP-GFP-LC3 vector for 24 h and then transfected with NC siRNA or SNAP-29 siRNA. After culturing in cisplatin (5 µg/mL) for 24 h, cells were imaged with a confocal microscope. Representative images of fluorescent LC3 puncta are shown. * P

    Journal: Theranostics

    Article Title: Down-regulation of OGT promotes cisplatin resistance by inducing autophagy in ovarian cancer

    doi: 10.7150/thno.27806

    Figure Lengend Snippet: The O -GlcNAcylation level of SNAP-29 is associated with autophagy activity induced by cisplatin. (A) Control and OGT-deficient SKOV3 cells were transfected with NC siRNA or SNAP-29 siRNA and then treated with cisplatin (5 µg/mL) for 24 h. The expression levels of LC3 and p62 were examined by western blotting. (B) OGT-deficient SKOV3 cells were transfected with mRFP-GFP-LC3 vector for 24 h and then transfected with NC siRNA or SNAP-29 siRNA. After culturing in cisplatin (5 µg/mL) for 24 h, cells were imaged with a confocal microscope. Representative images of fluorescent LC3 puncta are shown. * P

    Article Snippet: Cells were blocked with 0.1% BSA for 1 h at room temperature and subsequently incubated with anti-LC3 (1:100, CST, 2775s) and Lamp1 (1:200, Abcam, ab25630) at 4 °C overnight.

    Techniques: Activity Assay, Transfection, Expressing, Western Blot, Plasmid Preparation, Microscopy

    ROCK1 is up-regulated with neuronal autophagosome accumulation in AD mice. (A) Representative ROCK1 (upper panels) and LC3 (lower panels) immunofluorescence in AD and normal mouse brain sections. (B) Representative immunohistochemical (IHC) micrographs of Aβ (6E10, upper panels) and Beclin1 (lower panels) immunofluorescence in AD and normal mouse brain sections. (C) Quantification of ROCK1 immunopositive neurons in AD mouse brain sections compared to control sections ( n = 4). (D) Quantification of Beclin1 immunopositive neurons in AD mouse brain sections compared to control sections ( n = 4). Data are expressed as mean ± SEM; ** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: ROCK1 Is Associated with Alzheimer’s Disease-Specific Plaques, as well as Enhances Autophagosome Formation But not Autophagic Aβ Clearance

    doi: 10.3389/fncel.2016.00253

    Figure Lengend Snippet: ROCK1 is up-regulated with neuronal autophagosome accumulation in AD mice. (A) Representative ROCK1 (upper panels) and LC3 (lower panels) immunofluorescence in AD and normal mouse brain sections. (B) Representative immunohistochemical (IHC) micrographs of Aβ (6E10, upper panels) and Beclin1 (lower panels) immunofluorescence in AD and normal mouse brain sections. (C) Quantification of ROCK1 immunopositive neurons in AD mouse brain sections compared to control sections ( n = 4). (D) Quantification of Beclin1 immunopositive neurons in AD mouse brain sections compared to control sections ( n = 4). Data are expressed as mean ± SEM; ** p

    Article Snippet: After blocking for 30 min with 5% normal donkey serum, neurons were incubated with rabbit anti-LC3 (1:100, CST) antibody or mouse anti-ROCK1 (1:500, Abcam) antibody overnight at 4°C.

    Techniques: Mouse Assay, Immunofluorescence, Immunohistochemistry

    ROCK1 increases autophagy via an interaction with Beclin1. (A) Increased ROCK1 CA levels induced an increase in the intracellular autophagosome formation and accumulation. SH-SY5Y cells (upper panels) and primary neurons (lower panels) were co-transfected with ROCK1 CA plasmid and mCherry-GFP-LC3B adenovirus (pcDNA was used as a control). Fluorescence microscopy was used to detect the formation of GFP-LC3 puncta after 24 h post-transfection. (B) Expression of Beclin1 following ROCK1 knockdown and ROCK1 CA over-expression. (C) Quantification of Beclin1 following ROCK1 knockdown and ROCK1 CA over-expression. (D) HEK293 cells transfected with ROCK1 CA plasmid and pcDNA. Forty eight hours post-transfection co-immunoprecipitation (CoIP) was performed with cell lysates by using ROCK1 crosslinked agarose and then blotted with antibodies. (E) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (F) Quantification of CoIP Beclin1 levels. (G) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (H) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (I) ROCK1 CA plasmid transfection reverses 3-methyladenine (3-MA) induced autophagy inhibition. Representative immunoblot of LC3 II/LC3 I. (J) Increased Aβ40 secretion after 3-MA treatment (48 h) by ELISA. Data are presented as Mean ± SEM, students’ t test, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: ROCK1 Is Associated with Alzheimer’s Disease-Specific Plaques, as well as Enhances Autophagosome Formation But not Autophagic Aβ Clearance

    doi: 10.3389/fncel.2016.00253

    Figure Lengend Snippet: ROCK1 increases autophagy via an interaction with Beclin1. (A) Increased ROCK1 CA levels induced an increase in the intracellular autophagosome formation and accumulation. SH-SY5Y cells (upper panels) and primary neurons (lower panels) were co-transfected with ROCK1 CA plasmid and mCherry-GFP-LC3B adenovirus (pcDNA was used as a control). Fluorescence microscopy was used to detect the formation of GFP-LC3 puncta after 24 h post-transfection. (B) Expression of Beclin1 following ROCK1 knockdown and ROCK1 CA over-expression. (C) Quantification of Beclin1 following ROCK1 knockdown and ROCK1 CA over-expression. (D) HEK293 cells transfected with ROCK1 CA plasmid and pcDNA. Forty eight hours post-transfection co-immunoprecipitation (CoIP) was performed with cell lysates by using ROCK1 crosslinked agarose and then blotted with antibodies. (E) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (F) Quantification of CoIP Beclin1 levels. (G) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (H) Representative immunoblot of LC3 II/LC3 I after ROCK1 CA over-expression and ROCK1 knockdown. (I) ROCK1 CA plasmid transfection reverses 3-methyladenine (3-MA) induced autophagy inhibition. Representative immunoblot of LC3 II/LC3 I. (J) Increased Aβ40 secretion after 3-MA treatment (48 h) by ELISA. Data are presented as Mean ± SEM, students’ t test, * p

    Article Snippet: After blocking for 30 min with 5% normal donkey serum, neurons were incubated with rabbit anti-LC3 (1:100, CST) antibody or mouse anti-ROCK1 (1:500, Abcam) antibody overnight at 4°C.

    Techniques: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Expressing, Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay, Inhibition, Enzyme-linked Immunosorbent Assay

    ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: ORM-induced autophagy is upstream of apoptosis and pro-death in nature. ( A ) PA-1 cells were transfected for siRNA against either LC3 or Beclin 1, two critical autophagy proteins and cellular levels of LC3-II and cPARP were observed as markers of autophagic and apoptotic activity. ( B) PA-1 cells were treated with ORM alone (IC 50 dose), or pre-treated with lysosomal fusion inhibitor chloroquine (CQ; 10 μM, 2 h) or chemical chaperone and UPR inhibitor 4-PBA (5 mM; 2 h) before ORM treatment, stained with Annexin V-FITC/PI and analyzed by flow cytometry to assess apoptotic cell death (lower right quadrant = %early apoptotic population). ( C ) Viability (percentage of control) of PA-1 and OVCAR-3 cells pre-treated with CQ or 4-PBA and treated with ORM for 48 h measured through SRB assay; ( D , E ) PA-1 cells were pre-treated with 2.5 mM 3-MA or with 20 μM z-VAD-fmk or with 20 μM z-DEVD-fmk followed by 24 h ORM treatment (3-MA, z-DEVD) or 48 h ORM treatment (zVAD). PARP and LC3-II levels were analysed by immunoblotting.

    Article Snippet: Cells were incubated with anti-LC3 antibody (1:100 in PBS-2%BSA; Cell Signaling) overnight at 4 °C,washed thrice with PBS, incubated with secondary antibody (1:250) for 1 h and stained with Hoechst 33342 before mounting in glass slides and imaging in a confocal microscope.

    Techniques: Transfection, Activity Assay, Staining, Flow Cytometry, Cytometry, Sulforhodamine B Assay

    ORM administration reduces tumor progression and promotes autophagy in vivo . Reduction in both ( A ) tumor volume, and, ( B ) tumor mass was achieved when ORM (50 mg/kg body weight) was administered for 4 weeks. ( C ) LC3-II induction as a marker for autophagy activity in tumors isolated from control (VC) and ORM-treated (ORM) mice.

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: ORM administration reduces tumor progression and promotes autophagy in vivo . Reduction in both ( A ) tumor volume, and, ( B ) tumor mass was achieved when ORM (50 mg/kg body weight) was administered for 4 weeks. ( C ) LC3-II induction as a marker for autophagy activity in tumors isolated from control (VC) and ORM-treated (ORM) mice.

    Article Snippet: Cells were incubated with anti-LC3 antibody (1:100 in PBS-2%BSA; Cell Signaling) overnight at 4 °C,washed thrice with PBS, incubated with secondary antibody (1:250) for 1 h and stained with Hoechst 33342 before mounting in glass slides and imaging in a confocal microscope.

    Techniques: In Vivo, Marker, Activity Assay, Isolation, Mouse Assay

    Upregulation of UPR proteins activate ER stress response, generation of reactive oxygen species and JNK activation, which in turn regulate autophagy. ( A ) PA-1 and OVCAR-3 cells were treated with ORM IC 50 dose for indicated time-points and immunoblotted for UPR sensor proteins PERK, ATF6 and IRE1 as well as ER chaperone GRP78/BiP, downstream transcription factor CHOP/GADD153 and eukaryotic translation initiation factor eIF2α, apart from phosphorylated JNK (p-JNK) levels. ( B ) densitometric analyses of data represented in ( A ). ( C ) PA-1 and OVCAR-3 cells were grown in confocal glass-bottom dishes, treated with IC 50 dose of ORM for 24 h followed by staining with CM-H 2 DCFDA, a live-cell ROS marker, and imaged in confocal microscope. ( D ) Quantification of fluorescence intensity of each cell from data such as ( B ) as described in Methods. ( E ) PA-1 cells were pre-treated with NAC (5 mM, 2 h) or Tiron (5 μM, 1 h), treated with ORM (24 h) and LC3 conversion was analysed by western blotting. ( F ) PA-1 and OVCAR-3 cells were pre-treated or not with 4-PBA (5 mM; 2 h) or 3-MA (2.5 mM; 2 h) before treating with IC 50 dose of ORM and immunoblotting for GRP78 and PERK for quantification of ER stress and LC3 for quantification of autophagy. ( G ) PA-1 cells were pre-treated or not with small-molecule JNK inhibitor SP600126 (SP6; 10 μM,1 h) before IC 50 ORM treatment and immunoblotted for LC3 as a marker of autophagy. ( E ) Scale bars = 10 μm.

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: Upregulation of UPR proteins activate ER stress response, generation of reactive oxygen species and JNK activation, which in turn regulate autophagy. ( A ) PA-1 and OVCAR-3 cells were treated with ORM IC 50 dose for indicated time-points and immunoblotted for UPR sensor proteins PERK, ATF6 and IRE1 as well as ER chaperone GRP78/BiP, downstream transcription factor CHOP/GADD153 and eukaryotic translation initiation factor eIF2α, apart from phosphorylated JNK (p-JNK) levels. ( B ) densitometric analyses of data represented in ( A ). ( C ) PA-1 and OVCAR-3 cells were grown in confocal glass-bottom dishes, treated with IC 50 dose of ORM for 24 h followed by staining with CM-H 2 DCFDA, a live-cell ROS marker, and imaged in confocal microscope. ( D ) Quantification of fluorescence intensity of each cell from data such as ( B ) as described in Methods. ( E ) PA-1 cells were pre-treated with NAC (5 mM, 2 h) or Tiron (5 μM, 1 h), treated with ORM (24 h) and LC3 conversion was analysed by western blotting. ( F ) PA-1 and OVCAR-3 cells were pre-treated or not with 4-PBA (5 mM; 2 h) or 3-MA (2.5 mM; 2 h) before treating with IC 50 dose of ORM and immunoblotting for GRP78 and PERK for quantification of ER stress and LC3 for quantification of autophagy. ( G ) PA-1 cells were pre-treated or not with small-molecule JNK inhibitor SP600126 (SP6; 10 μM,1 h) before IC 50 ORM treatment and immunoblotted for LC3 as a marker of autophagy. ( E ) Scale bars = 10 μm.

    Article Snippet: Cells were incubated with anti-LC3 antibody (1:100 in PBS-2%BSA; Cell Signaling) overnight at 4 °C,washed thrice with PBS, incubated with secondary antibody (1:250) for 1 h and stained with Hoechst 33342 before mounting in glass slides and imaging in a confocal microscope.

    Techniques: Activation Assay, Staining, Marker, Microscopy, Fluorescence, Western Blot

    ORM induces autophagic flux in ovarian cancer cells PA-1 and OVCAR-3. ( A ) Dose-response curve of ORM in ovarian cancer cell lines PA-1 and OVCAR-3. ( B ) PA-1 cells were stained with MDC, a marker of acidic cellular compartments after treatment with indicated time periods with IC 50 dose of ORM. ( C ) quantification of MDC-positive dots from ( B ) as described in Methods. ( D ) Confocal microscopy images of PA-1 and OVCAR-3 cells treated with IC 50 dose of ORM and immunostained with LC3 (an autophagosome marker). ( E ) PA-1 cells were pre-treated with or without 3-MA (class III PI3K inhibitor; 2.5 mM) or CQ (lysosomal fusion inhibitor; 10 μM), immunostained with LC3 and imaged in confocal microscope. ( F ) Quantification of LC3 puncta from data such as ( E ) as described in Methods. ( G ) PA-1 and OVCAR-3 cells were treated with ORM (IC 50 dose) for indicated time-points and probed for autophagy markers LC3 and Beclin 1, quantified in ( H ). ( I ) PA-1 and OVCAR-3 cells were pre-treated or not with lysosomal fusion inhibitor BafA1 (100 nM; 2 h) to inhibit fusion of autophagosomes with lysosomes. LC3-II conversion was observed with immunoblotting and quantified. ( J ) PA-1 cells were transfected with tfLC3 (as described in Methods), treated with ORM for 24 h and imaged in confocal microscope. Scale bars = 20 μm ( B ); 10 μm ( D ).

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: ORM induces autophagic flux in ovarian cancer cells PA-1 and OVCAR-3. ( A ) Dose-response curve of ORM in ovarian cancer cell lines PA-1 and OVCAR-3. ( B ) PA-1 cells were stained with MDC, a marker of acidic cellular compartments after treatment with indicated time periods with IC 50 dose of ORM. ( C ) quantification of MDC-positive dots from ( B ) as described in Methods. ( D ) Confocal microscopy images of PA-1 and OVCAR-3 cells treated with IC 50 dose of ORM and immunostained with LC3 (an autophagosome marker). ( E ) PA-1 cells were pre-treated with or without 3-MA (class III PI3K inhibitor; 2.5 mM) or CQ (lysosomal fusion inhibitor; 10 μM), immunostained with LC3 and imaged in confocal microscope. ( F ) Quantification of LC3 puncta from data such as ( E ) as described in Methods. ( G ) PA-1 and OVCAR-3 cells were treated with ORM (IC 50 dose) for indicated time-points and probed for autophagy markers LC3 and Beclin 1, quantified in ( H ). ( I ) PA-1 and OVCAR-3 cells were pre-treated or not with lysosomal fusion inhibitor BafA1 (100 nM; 2 h) to inhibit fusion of autophagosomes with lysosomes. LC3-II conversion was observed with immunoblotting and quantified. ( J ) PA-1 cells were transfected with tfLC3 (as described in Methods), treated with ORM for 24 h and imaged in confocal microscope. Scale bars = 20 μm ( B ); 10 μm ( D ).

    Article Snippet: Cells were incubated with anti-LC3 antibody (1:100 in PBS-2%BSA; Cell Signaling) overnight at 4 °C,washed thrice with PBS, incubated with secondary antibody (1:250) for 1 h and stained with Hoechst 33342 before mounting in glass slides and imaging in a confocal microscope.

    Techniques: Staining, Marker, Confocal Microscopy, Microscopy, Transfection

    The Akt/mTOR pathway is involved in ORM-induced autophagy. ( A ) PA-1 and OVCAR-3 cells were treated with ORM IC 50 dose for indicated time-points and immunoblotted for mTOR pathway proteins mTOR (phosphorylation sites at Ser2448 and Ser2481), Akt (phosphorylation sites at Ser473 and Thr308) and p70 S6k (at Thr389; in PA-1). ( B ) densitometric analyses for p-Akt (Ser473) and p-mTOR (Ser2448) from data represented in ( A ). ( C ) PA-1 and OVCAR-3 cells were treated with ORM alone or in combination with Akti (inhibitor of Akt 1/2; 10 μM) for 24 h and cellular levels of p-mTOR (as a downstream regulator of autophagy) and LC3 were observed by immunoblotting.

    Journal: Scientific Reports

    Article Title: Ormeloxifene-induced unfolded protein response contributes to autophagy-associated apoptosis via disruption of Akt/mTOR and activation of JNK

    doi: 10.1038/s41598-018-20541-8

    Figure Lengend Snippet: The Akt/mTOR pathway is involved in ORM-induced autophagy. ( A ) PA-1 and OVCAR-3 cells were treated with ORM IC 50 dose for indicated time-points and immunoblotted for mTOR pathway proteins mTOR (phosphorylation sites at Ser2448 and Ser2481), Akt (phosphorylation sites at Ser473 and Thr308) and p70 S6k (at Thr389; in PA-1). ( B ) densitometric analyses for p-Akt (Ser473) and p-mTOR (Ser2448) from data represented in ( A ). ( C ) PA-1 and OVCAR-3 cells were treated with ORM alone or in combination with Akti (inhibitor of Akt 1/2; 10 μM) for 24 h and cellular levels of p-mTOR (as a downstream regulator of autophagy) and LC3 were observed by immunoblotting.

    Article Snippet: Cells were incubated with anti-LC3 antibody (1:100 in PBS-2%BSA; Cell Signaling) overnight at 4 °C,washed thrice with PBS, incubated with secondary antibody (1:250) for 1 h and stained with Hoechst 33342 before mounting in glass slides and imaging in a confocal microscope.

    Techniques:

    Berberine induces neuronal autophagy. (A) Autophagy activation was observed by a transmission electron microscope without or with berberine for 1, 2, and 4 h. (B and C) Accumulation of LC3-positive puncta (green) in neurons was visualized by immunofluorescence

    Journal: American Journal of Translational Research

    Article Title: Pharmacologic preconditioning with berberine attenuating ischemia-induced apoptosis and promoting autophagy in neuron

    doi:

    Figure Lengend Snippet: Berberine induces neuronal autophagy. (A) Autophagy activation was observed by a transmission electron microscope without or with berberine for 1, 2, and 4 h. (B and C) Accumulation of LC3-positive puncta (green) in neurons was visualized by immunofluorescence

    Article Snippet: The formalin-fixed neurons were incubated with rabbit anti-LC3 antibody (diluted 1:100; CST, Boston, MA) overnight at 4°C.

    Techniques: Activation Assay, Transmission Assay, Microscopy, Immunofluorescence

    MHCD ameliorates Doxorubicin-induced increases in autophagy by inhibiting LC3 and beclin-1 protein levels. (A) The expression of LC3 and beclin-1 in the glomeruli of Doxorubicin-induced nephrotic rats was assessed using immunohistochemistry (magnification, ×400). Quantification of (B) beclin-1 and (C) LC3. Data are expressed the mean ± standard deviation (n=10). *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Modified Huangqi Chifeng decoction inhibits excessive autophagy to protect against Doxorubicin-induced nephrotic syndrome in rats via the PI3K/mTOR signaling pathway

    doi: 10.3892/etm.2018.6492

    Figure Lengend Snippet: MHCD ameliorates Doxorubicin-induced increases in autophagy by inhibiting LC3 and beclin-1 protein levels. (A) The expression of LC3 and beclin-1 in the glomeruli of Doxorubicin-induced nephrotic rats was assessed using immunohistochemistry (magnification, ×400). Quantification of (B) beclin-1 and (C) LC3. Data are expressed the mean ± standard deviation (n=10). *P

    Article Snippet: Anti-beclin1 (1:1,000; cat. no. ab210498), anti-LC3 (1:1,000; cat. no. ab48394), anti-PI3K (1:1,000; cat. no. ab86714) and anti-mTOR (1:1,000; cat. no. ab2732) primary antibodies were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Immunohistochemistry, Standard Deviation

    MHCD decreases the expression of autophagy signaling pathway proteins in rats with Doxorubicin-induced nephrosis. LC3-I, LC3II, beclin-1, PI3K and mTOR expression was (A) measured using western blotting and (B) quantified. Data are expressed as the mean ± standard deviation. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Modified Huangqi Chifeng decoction inhibits excessive autophagy to protect against Doxorubicin-induced nephrotic syndrome in rats via the PI3K/mTOR signaling pathway

    doi: 10.3892/etm.2018.6492

    Figure Lengend Snippet: MHCD decreases the expression of autophagy signaling pathway proteins in rats with Doxorubicin-induced nephrosis. LC3-I, LC3II, beclin-1, PI3K and mTOR expression was (A) measured using western blotting and (B) quantified. Data are expressed as the mean ± standard deviation. *P

    Article Snippet: Anti-beclin1 (1:1,000; cat. no. ab210498), anti-LC3 (1:1,000; cat. no. ab48394), anti-PI3K (1:1,000; cat. no. ab86714) and anti-mTOR (1:1,000; cat. no. ab2732) primary antibodies were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Standard Deviation

    The neuroprotective effect of bFGF after global cerebral I/R functioned by restraining excessive autophagy via the mTOR pathway. a Immunoblots of LC3, p62, Beclin-1, mTOR, and p-mTOR was assessed with β-actin as a loading control. b-e Densitometric analysis (mean ± SEM, n = 3 animals per group) of the proteins from (A) normalized to the respective loading controls. f Representative immunofluorescence images of LC3 (green) and DAPI (blue) double staining in medial CA1 region 24 h after I/R. g Quantitative analysis of LC3B puncta per cell. More than 30 cells per condition were included. The data are presented as the mean ± SEM. *** P

    Journal: Cell Death & Disease

    Article Title: bFGF plays a neuroprotective role by suppressing excessive autophagy and apoptosis after transient global cerebral ischemia in rats

    doi: 10.1038/s41419-017-0229-7

    Figure Lengend Snippet: The neuroprotective effect of bFGF after global cerebral I/R functioned by restraining excessive autophagy via the mTOR pathway. a Immunoblots of LC3, p62, Beclin-1, mTOR, and p-mTOR was assessed with β-actin as a loading control. b-e Densitometric analysis (mean ± SEM, n = 3 animals per group) of the proteins from (A) normalized to the respective loading controls. f Representative immunofluorescence images of LC3 (green) and DAPI (blue) double staining in medial CA1 region 24 h after I/R. g Quantitative analysis of LC3B puncta per cell. More than 30 cells per condition were included. The data are presented as the mean ± SEM. *** P

    Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-GAPDH (1:1000, #5174), rabbit anti-p-mTOR (1:1000, #2974), rabbit anti-mTOR (1:1000, #2983), rabbit anti-cleaved Caspase-3 (1:250, #9664), mouse anti-p53 (1:1000, #2524), rabbit anti-Puma (1:200, #7467), rabbit anti-p-4E-BP1 (1:1000, #2855), rabbit anti-4E-BP1 (#9644), rabbit anti-p-p70 S6 (1:1000, #9234), rabbit anti-p70 S6 (1:1000, #2708), rabbit anti-p-ULK1 (1:1000, 14202), rabbit anti-ULK1 (1:1000, #8054), and rabbit anti-COX IV (1:1000, #4850) from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-LC3 (1:2000, ab51520), rabbit anti-p62 (1:1000, ab109012), rabbit anti-cytochrome c (1:2000, ab133504), and rabbit anti-Bax (1:800, ab32503) from Abcam (Cambridge, MA, USA); mouse anti-bFGF (1:1000, sc365106), rabbit anti-Beclin 1 (1:1000, sc-11427), and rabbit anti-Bcl-2 (1:1500, sc-783) from Santa Cruz Biotechnology, Inc. (Santa Cruz CA, USA); and mouse anti-β-actin (1:1000, EM21002), goat anti-rabbit secondary antibody (1:5000, HA1001-100), and goat anti-mouse secondary antibody (1:4000, HA1006) from Hangzhou Huaan Biotechnology (Hangzhou, China).

    Techniques: Western Blot, Immunofluorescence, Double Staining

    The effects of miR-24-3p and BNIP3 on mitophagy-related protein expression levels and U87-GSC and U251-GSC malignant behavior. (A,B) Western blot analysis of the effects of miR-24-3p and BNIP3 co-transfection on LC3-II/I, P62, TOMM 20, and CYPD expression levels. The LC3-II/I ratio was decreased, but P62, TOMM20 and CYPD expression levels were increased in the Agomir-24-3p+BNIP3(–) group. Data are presented as the mean ± SD of n = 3. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Endothelial Monocyte-Activating Polypeptide-II Induces BNIP3-Mediated Mitophagy to Enhance Temozolomide Cytotoxicity of Glioma Stem Cells via Down-Regulating MiR-24-3p

    doi: 10.3389/fnmol.2018.00092

    Figure Lengend Snippet: The effects of miR-24-3p and BNIP3 on mitophagy-related protein expression levels and U87-GSC and U251-GSC malignant behavior. (A,B) Western blot analysis of the effects of miR-24-3p and BNIP3 co-transfection on LC3-II/I, P62, TOMM 20, and CYPD expression levels. The LC3-II/I ratio was decreased, but P62, TOMM20 and CYPD expression levels were increased in the Agomir-24-3p+BNIP3(–) group. Data are presented as the mean ± SD of n = 3. * P

    Article Snippet: The proteins (30–60 μg) were separated by 10–15% SDS-PAGE and transferred onto PVDF membranes, which were blocked with 5% non-fat milk for 2 h and then incubated overnight with primary antibodies against LC3 (1:1000, Abcam, Cambridge, MA, USA, ab51520), P62/SQSTM1 (1:1000, Proteintech, Chicago, IL, USA, ab56416), GAPDH(1:10,000, Proteintech, Chicago, IL, USA, 6000-4-Ig), BNIP3 (1:1000, Abclonal, Cambridge, MA, USA, A5683), TOMM20 (1:1000, Proteintech, Chicago, IL, USA, 11802-1-AP), and CYPD (1:1000, Proteintech, Chicago, IL, USA, 12716-1-AP).

    Techniques: Expressing, Western Blot, Cotransfection

    The effects of EMAP-II and TMZ on mitophagy in U87-GSCs and U251-GSCs. (A) Representative TEM image of autophagosomes in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ. Autophagysome activity levels were gradually increased in the EMAP-II and TMZ groups and the EMAP-II and TMZ combination group. Scale bar corresponds to 1 μm. (B,C) The distribution and expression of LC3 in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ were analyzed by immunofluorescence assay with Lyso-Tracker and Mito-Tracker. The expression of LC3 indicates the occurrence of autophagy. The distribution of LC3 that overlap with lysosome or mitochondria indicates autophagy or mitophagy. Scale bars represent 20 μm. (D) Western blot analysis of the LC3-II/I ratio and P62, TOMM 20, and CYPD expression levels in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ. The LC3-II/I ratio increased, but P62, TOMM 20, and CYPD expression decreased in GSCs treated with EMAP-II and TMZ. Data are presented as the mean ± SD of n = 3. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Endothelial Monocyte-Activating Polypeptide-II Induces BNIP3-Mediated Mitophagy to Enhance Temozolomide Cytotoxicity of Glioma Stem Cells via Down-Regulating MiR-24-3p

    doi: 10.3389/fnmol.2018.00092

    Figure Lengend Snippet: The effects of EMAP-II and TMZ on mitophagy in U87-GSCs and U251-GSCs. (A) Representative TEM image of autophagosomes in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ. Autophagysome activity levels were gradually increased in the EMAP-II and TMZ groups and the EMAP-II and TMZ combination group. Scale bar corresponds to 1 μm. (B,C) The distribution and expression of LC3 in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ were analyzed by immunofluorescence assay with Lyso-Tracker and Mito-Tracker. The expression of LC3 indicates the occurrence of autophagy. The distribution of LC3 that overlap with lysosome or mitochondria indicates autophagy or mitophagy. Scale bars represent 20 μm. (D) Western blot analysis of the LC3-II/I ratio and P62, TOMM 20, and CYPD expression levels in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ. The LC3-II/I ratio increased, but P62, TOMM 20, and CYPD expression decreased in GSCs treated with EMAP-II and TMZ. Data are presented as the mean ± SD of n = 3. * P

    Article Snippet: The proteins (30–60 μg) were separated by 10–15% SDS-PAGE and transferred onto PVDF membranes, which were blocked with 5% non-fat milk for 2 h and then incubated overnight with primary antibodies against LC3 (1:1000, Abcam, Cambridge, MA, USA, ab51520), P62/SQSTM1 (1:1000, Proteintech, Chicago, IL, USA, ab56416), GAPDH(1:10,000, Proteintech, Chicago, IL, USA, 6000-4-Ig), BNIP3 (1:1000, Abclonal, Cambridge, MA, USA, A5683), TOMM20 (1:1000, Proteintech, Chicago, IL, USA, 11802-1-AP), and CYPD (1:1000, Proteintech, Chicago, IL, USA, 12716-1-AP).

    Techniques: Transmission Electron Microscopy, Activity Assay, Expressing, Immunofluorescence, Western Blot

    The effects of BNIP3 on mitophagy-related protein expression levels in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ. (A) Western blot analysis of the effects of BNIP3 overexpression and silencing on the LC3-II/I ratio and BNIP3, P62, TOMM 20, and CYPD expression levels. BNIP3 overexpression up-regulated LC3-II/I ratio and reduced P62, TOMM 20, and CYPD expression. The Data are presented as the mean ± SD of n = 3. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Endothelial Monocyte-Activating Polypeptide-II Induces BNIP3-Mediated Mitophagy to Enhance Temozolomide Cytotoxicity of Glioma Stem Cells via Down-Regulating MiR-24-3p

    doi: 10.3389/fnmol.2018.00092

    Figure Lengend Snippet: The effects of BNIP3 on mitophagy-related protein expression levels in U87-GSCs and U251-GSCs treated with EMAP-II and TMZ. (A) Western blot analysis of the effects of BNIP3 overexpression and silencing on the LC3-II/I ratio and BNIP3, P62, TOMM 20, and CYPD expression levels. BNIP3 overexpression up-regulated LC3-II/I ratio and reduced P62, TOMM 20, and CYPD expression. The Data are presented as the mean ± SD of n = 3. * P

    Article Snippet: The proteins (30–60 μg) were separated by 10–15% SDS-PAGE and transferred onto PVDF membranes, which were blocked with 5% non-fat milk for 2 h and then incubated overnight with primary antibodies against LC3 (1:1000, Abcam, Cambridge, MA, USA, ab51520), P62/SQSTM1 (1:1000, Proteintech, Chicago, IL, USA, ab56416), GAPDH(1:10,000, Proteintech, Chicago, IL, USA, 6000-4-Ig), BNIP3 (1:1000, Abclonal, Cambridge, MA, USA, A5683), TOMM20 (1:1000, Proteintech, Chicago, IL, USA, 11802-1-AP), and CYPD (1:1000, Proteintech, Chicago, IL, USA, 12716-1-AP).

    Techniques: Expressing, Western Blot, Over Expression

    The expression of LC3II and p62 in left ventricle. The protein levels of LC3II and p62 were quantified by Western blotting in the methods (a and c). Left ventricle sections stained with an anti-LC3 antibody (b). Original magnification: ×200. Bar, 100 μm. Mean ± standard deviation, six rats per group, * P

    Journal: Chinese Medical Journal

    Article Title: Accelerated Autophagy of Cecal Ligation and Puncture-Induced Myocardial Dysfunction and Its Correlation with Mammalian Target of Rapamycin Pathway in Rats

    doi: 10.4103/0366-6999.231522

    Figure Lengend Snippet: The expression of LC3II and p62 in left ventricle. The protein levels of LC3II and p62 were quantified by Western blotting in the methods (a and c). Left ventricle sections stained with an anti-LC3 antibody (b). Original magnification: ×200. Bar, 100 μm. Mean ± standard deviation, six rats per group, * P

    Article Snippet: [ ] The heart sections were immune with anti-LC3 antibody (ab48394, Abcam, 1:1000 dilution).

    Techniques: Expressing, Western Blot, Staining, Standard Deviation

    TNP-1 induced autophagy in a shape- and composition-dependent manner. a Fluorescent microscopy images of EGFP-LC3/HeLa cells treated with PBS (control) or 10 µg mL −1 of CuPd nanoparticles for 24 h. Scale bar, 10 µm. The right panel shows the quantified results for the percentage of cells containing at least 5 EGFP-LC3 dots. Mean ± s.e.m. n = 5. *** p

    Journal: Nature Communications

    Article Title: Harnessing copper-palladium alloy tetrapod nanoparticle-induced pro-survival autophagy for optimized photothermal therapy of drug-resistant cancer

    doi: 10.1038/s41467-018-06529-y

    Figure Lengend Snippet: TNP-1 induced autophagy in a shape- and composition-dependent manner. a Fluorescent microscopy images of EGFP-LC3/HeLa cells treated with PBS (control) or 10 µg mL −1 of CuPd nanoparticles for 24 h. Scale bar, 10 µm. The right panel shows the quantified results for the percentage of cells containing at least 5 EGFP-LC3 dots. Mean ± s.e.m. n = 5. *** p

    Article Snippet: Anti-LC3 antibody (NB100–2220, 1:2000 dilution) were purchased from Novus Biologicals.

    Techniques: Microscopy

    TNP-1 induced complete autophagy in a ROS-dependent fashion. a Intracellular ROS detected with DCFH-DA and analyzed by FACS after treatment with PBS (control) or TNPs (10 µg mL −1 ) for 24 h. b FACS analysis of intracellular ROS after HeLa cells were treated with PBS (control), TNP-1 (10 µg mL −1 ), or TNP-1 in the presence of MitoTempo (M; 0.5 mM) or VAS 2870 (V; 20 μM) for 24 h. c Western blotting of HeLa cells with LC3 and GAPDH antibodies after the indicated treatment for 24 h. The right panel showed quantified results. MT, MitoTemper; VAS, VAS 2870. Mean ± s.e.m. n = 3. *** p

    Journal: Nature Communications

    Article Title: Harnessing copper-palladium alloy tetrapod nanoparticle-induced pro-survival autophagy for optimized photothermal therapy of drug-resistant cancer

    doi: 10.1038/s41467-018-06529-y

    Figure Lengend Snippet: TNP-1 induced complete autophagy in a ROS-dependent fashion. a Intracellular ROS detected with DCFH-DA and analyzed by FACS after treatment with PBS (control) or TNPs (10 µg mL −1 ) for 24 h. b FACS analysis of intracellular ROS after HeLa cells were treated with PBS (control), TNP-1 (10 µg mL −1 ), or TNP-1 in the presence of MitoTempo (M; 0.5 mM) or VAS 2870 (V; 20 μM) for 24 h. c Western blotting of HeLa cells with LC3 and GAPDH antibodies after the indicated treatment for 24 h. The right panel showed quantified results. MT, MitoTemper; VAS, VAS 2870. Mean ± s.e.m. n = 3. *** p

    Article Snippet: Anti-LC3 antibody (NB100–2220, 1:2000 dilution) were purchased from Novus Biologicals.

    Techniques: FACS, Western Blot

    Schematic model depicting the IFN-γ–mediated LAP-like process against liver-stage P. vivax . P. vivax sporozoites actively invade human hepatocytes and then reside within the parasitophorous vacuoles. IFN-γ treatment leads to the decoration of the parasitophorous vacuole membrane (PVM) with host LC3, in a process involving Beclin 1, PI3K, and Atg5. The LC3-decorated PVMs then fuse with lysosomes, which kills the parasite.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    doi: 10.1073/pnas.1525606113

    Figure Lengend Snippet: Schematic model depicting the IFN-γ–mediated LAP-like process against liver-stage P. vivax . P. vivax sporozoites actively invade human hepatocytes and then reside within the parasitophorous vacuoles. IFN-γ treatment leads to the decoration of the parasitophorous vacuole membrane (PVM) with host LC3, in a process involving Beclin 1, PI3K, and Atg5. The LC3-decorated PVMs then fuse with lysosomes, which kills the parasite.

    Article Snippet: The polyclonal antibodies used in the IFAs were anti-LC3 (1:500; MBL International) and Alexa Fluor 647-conjugated anti-LC3 (1:250; Novus Biologicals).

    Techniques:

    IFN-γ–induced liver-stage P. vivax elimination depends on Beclin 1 and PI3K. ( A–C ) HC04 cells were transfected with scramble control siRNAs or siRNAs against Beclin 1 and cDNAs encoding RFP-GFP-LC3. At 48 h posttransfection, the cells were treated or not with IFN-γ for 4 h and then processed for fluorescence microscopy. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified, and the percentage of puncta-containing cells was determined by analyzing at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also quantified in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM relative to the full control. N.S., not significant. (Scale bar: 5 µm.) ( D ) Beclin 1 can be successfully depleted in HC04 cells. HC04 cells were transfected with scramble control siRNAs or siRNAs against Beclin 1 and then harvested at 48 h posttransfection for immunoblot analysis. Actin served as an internal loading control. ( E and F ) Beclin 1-deficient or -proficient HC04 cells were infected with P. vivax sporozoites at an MOI of 1, followed by IFN-γ treatment for 4 h. The cells were washed and then maintained in complete medium until being processed for IFAs on day 4. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.) ( G and H ) HC04 cells infected with P. vivax sporozoites were treated with IFN-γ with or without the PI3K inhibitor 3-MA for 4 h, washed, and maintained in complete medium until being processed for IFA on day 4. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    doi: 10.1073/pnas.1525606113

    Figure Lengend Snippet: IFN-γ–induced liver-stage P. vivax elimination depends on Beclin 1 and PI3K. ( A–C ) HC04 cells were transfected with scramble control siRNAs or siRNAs against Beclin 1 and cDNAs encoding RFP-GFP-LC3. At 48 h posttransfection, the cells were treated or not with IFN-γ for 4 h and then processed for fluorescence microscopy. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified, and the percentage of puncta-containing cells was determined by analyzing at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also quantified in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM relative to the full control. N.S., not significant. (Scale bar: 5 µm.) ( D ) Beclin 1 can be successfully depleted in HC04 cells. HC04 cells were transfected with scramble control siRNAs or siRNAs against Beclin 1 and then harvested at 48 h posttransfection for immunoblot analysis. Actin served as an internal loading control. ( E and F ) Beclin 1-deficient or -proficient HC04 cells were infected with P. vivax sporozoites at an MOI of 1, followed by IFN-γ treatment for 4 h. The cells were washed and then maintained in complete medium until being processed for IFAs on day 4. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.) ( G and H ) HC04 cells infected with P. vivax sporozoites were treated with IFN-γ with or without the PI3K inhibitor 3-MA for 4 h, washed, and maintained in complete medium until being processed for IFA on day 4. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.)

    Article Snippet: The polyclonal antibodies used in the IFAs were anti-LC3 (1:500; MBL International) and Alexa Fluor 647-conjugated anti-LC3 (1:250; Novus Biologicals).

    Techniques: Transfection, Fluorescence, Microscopy, Infection, Immunofluorescence

    Anti-LC3 antibodies and LTR do not recognize isolated P. vivax sporozoites. ( A–C ) Isolated P. vivax sporozoites were spotted onto a 10-well antigen slide, fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin/3% BSA in PBS. The sporozoites were then stained with Alexa Fluor 647-conjugated rabbit anti-LC3 antibody at room temperature for 2 h ( A ). Alternatively, the sporozoites were stained with rabbit anti-LC3 antibody at 4 °C overnight, followed by Alexa Fluor 568-conjugated anti-rabbit secondary antibody at room temperature for 2 h ( B ) or with Alexa Fluor 568-conjugated anti-rabbit secondary antibody alone ( C ). The samples were then mounted and analyzed by confocal microscopy. ( D ) Isolated P. vivax sporozoites were incubated with LTR dye for 1 h at room temperature, washed twice with PBS, and spotted onto a 10-well antigen slide. The mounted samples were analyzed by confocal microscopy. Sporozoites are marked with white arrowheads. (Scale bar: 5 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    doi: 10.1073/pnas.1525606113

    Figure Lengend Snippet: Anti-LC3 antibodies and LTR do not recognize isolated P. vivax sporozoites. ( A–C ) Isolated P. vivax sporozoites were spotted onto a 10-well antigen slide, fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin/3% BSA in PBS. The sporozoites were then stained with Alexa Fluor 647-conjugated rabbit anti-LC3 antibody at room temperature for 2 h ( A ). Alternatively, the sporozoites were stained with rabbit anti-LC3 antibody at 4 °C overnight, followed by Alexa Fluor 568-conjugated anti-rabbit secondary antibody at room temperature for 2 h ( B ) or with Alexa Fluor 568-conjugated anti-rabbit secondary antibody alone ( C ). The samples were then mounted and analyzed by confocal microscopy. ( D ) Isolated P. vivax sporozoites were incubated with LTR dye for 1 h at room temperature, washed twice with PBS, and spotted onto a 10-well antigen slide. The mounted samples were analyzed by confocal microscopy. Sporozoites are marked with white arrowheads. (Scale bar: 5 µm.)

    Article Snippet: The polyclonal antibodies used in the IFAs were anti-LC3 (1:500; MBL International) and Alexa Fluor 647-conjugated anti-LC3 (1:250; Novus Biologicals).

    Techniques: Isolation, Staining, Confocal Microscopy, Incubation

    ULK1 is dispensable for the killing of liver-stage P. vivax mediated by IFN-γ. ( A–C ) ULK1 is not required for IFN-γ–induced LC3 puncta formation in HC04 cells. The cells were cotransfected with cDNAs encoding RFP-GFP-LC3 and either siRNAs against ULK1 or scramble control siRNAs. At 48 h posttransfection, they were treated with IFN-γ for 4 h and then processed for fluorescence microscopy analysis. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified by determining the percentage of puncta-containing cells among at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also examined in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM; the results are expressed relative to the full control. (Scale bar: 5 µm.) ( D ) ULK1 immunoblot analysis after depletion of the protein. HC04 cells were transfected with either scramble control siRNAs or siRNAs against ULK1. At 48 h posttransfection, they were harvested for Western blot analysis. ( E and F ) ULK1-deficient or control HC04 cells were infected with P. vivax sporozoites at an MOI of 1, treated with IFN-γ for 4 h, washed, and then maintained in complete medium until being harvested on day 4 for IFA. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. N.S., not significant. (Scale bar: 5 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    doi: 10.1073/pnas.1525606113

    Figure Lengend Snippet: ULK1 is dispensable for the killing of liver-stage P. vivax mediated by IFN-γ. ( A–C ) ULK1 is not required for IFN-γ–induced LC3 puncta formation in HC04 cells. The cells were cotransfected with cDNAs encoding RFP-GFP-LC3 and either siRNAs against ULK1 or scramble control siRNAs. At 48 h posttransfection, they were treated with IFN-γ for 4 h and then processed for fluorescence microscopy analysis. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified by determining the percentage of puncta-containing cells among at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also examined in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM; the results are expressed relative to the full control. (Scale bar: 5 µm.) ( D ) ULK1 immunoblot analysis after depletion of the protein. HC04 cells were transfected with either scramble control siRNAs or siRNAs against ULK1. At 48 h posttransfection, they were harvested for Western blot analysis. ( E and F ) ULK1-deficient or control HC04 cells were infected with P. vivax sporozoites at an MOI of 1, treated with IFN-γ for 4 h, washed, and then maintained in complete medium until being harvested on day 4 for IFA. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. N.S., not significant. (Scale bar: 5 µm.)

    Article Snippet: The polyclonal antibodies used in the IFAs were anti-LC3 (1:500; MBL International) and Alexa Fluor 647-conjugated anti-LC3 (1:250; Novus Biologicals).

    Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Infection, Immunofluorescence

    Liver-stage P. vivax colocalization with LC3 and LTR + vesicles increases in response to IFN-γ. ( A and B ) HC04 cells were infected with P. vivax sporozoites at an MOI of 1, treated with IFN-γ for 4 h, and then processed for confocal microscopy analysis of the colocalization of liver-stage P. vivax (labeled with CSP) and LC3. Data are mean ± SEM from at least three independent experiments. At least 100 liver-stage P. vivax parasites were quantified per condition. The results are expressed relative to the full control. ( C and D ) HC04 cells were infected with P. vivax sporozoites as in A and B and then incubated in complete medium containing LTR to stain acidic compartments in the presence or absence of IFN-γ treatment for 4 h. Cells were then processed for confocal microscopy analysis of the colocalization of liver-stage P. vivax (labeled with CSP) and LTR + vesicles. Data are mean ± SEM of at least three independent experiments. At least 100 liver-stage P. vivax parasites per condition were quantified. The results are expressed relative to the full control. ( E and F ) HC04 cells were infected with P. vivax sporozoites at an MOI of 1, treated with IFN-γ for 4 h, and processed for confocal microscopy analysis for the colocalization of the PVM of UIS4-labeled P. vivax and LC3. Data are mean ± SEM of at least three independent experiments. At least 50 parasites per condition were quantified. The results are expressed relative to the full control. ( G and H ) HC04 cells were infected with P. vivax sporozoites and then incubated in complete medium containing LTR to stain acidic compartments in the presence or absence of IFN-γ treatment for 4 h. Cells were then processed for confocal microscopy analysis for the colocalization of the UIS4-labeled PVM and LTR + vesicles. Data are mean ± SEM of at least three independent experiments. At least 50 parasites per condition were quantified. The results are expressed relative to the full control. (Scale bar: 5 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    doi: 10.1073/pnas.1525606113

    Figure Lengend Snippet: Liver-stage P. vivax colocalization with LC3 and LTR + vesicles increases in response to IFN-γ. ( A and B ) HC04 cells were infected with P. vivax sporozoites at an MOI of 1, treated with IFN-γ for 4 h, and then processed for confocal microscopy analysis of the colocalization of liver-stage P. vivax (labeled with CSP) and LC3. Data are mean ± SEM from at least three independent experiments. At least 100 liver-stage P. vivax parasites were quantified per condition. The results are expressed relative to the full control. ( C and D ) HC04 cells were infected with P. vivax sporozoites as in A and B and then incubated in complete medium containing LTR to stain acidic compartments in the presence or absence of IFN-γ treatment for 4 h. Cells were then processed for confocal microscopy analysis of the colocalization of liver-stage P. vivax (labeled with CSP) and LTR + vesicles. Data are mean ± SEM of at least three independent experiments. At least 100 liver-stage P. vivax parasites per condition were quantified. The results are expressed relative to the full control. ( E and F ) HC04 cells were infected with P. vivax sporozoites at an MOI of 1, treated with IFN-γ for 4 h, and processed for confocal microscopy analysis for the colocalization of the PVM of UIS4-labeled P. vivax and LC3. Data are mean ± SEM of at least three independent experiments. At least 50 parasites per condition were quantified. The results are expressed relative to the full control. ( G and H ) HC04 cells were infected with P. vivax sporozoites and then incubated in complete medium containing LTR to stain acidic compartments in the presence or absence of IFN-γ treatment for 4 h. Cells were then processed for confocal microscopy analysis for the colocalization of the UIS4-labeled PVM and LTR + vesicles. Data are mean ± SEM of at least three independent experiments. At least 50 parasites per condition were quantified. The results are expressed relative to the full control. (Scale bar: 5 µm.)

    Article Snippet: The polyclonal antibodies used in the IFAs were anti-LC3 (1:500; MBL International) and Alexa Fluor 647-conjugated anti-LC3 (1:250; Novus Biologicals).

    Techniques: Infection, Confocal Microscopy, Labeling, Incubation, Staining

    IFN-γ treatment increases LC3 puncta formation in HC04 cells. ( A–C ) HC04 cells were transfected with cDNAs encoding RFP-GFP-LC3. At 48 h posttransfection, they were treated with IFN-γ for 4 h in the presence or absence of 3-MA. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified by determining the percentage of puncta-containing cells among at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also examined in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM. The results are expressed relative to the full control. (Scale bar: 5 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    doi: 10.1073/pnas.1525606113

    Figure Lengend Snippet: IFN-γ treatment increases LC3 puncta formation in HC04 cells. ( A–C ) HC04 cells were transfected with cDNAs encoding RFP-GFP-LC3. At 48 h posttransfection, they were treated with IFN-γ for 4 h in the presence or absence of 3-MA. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified by determining the percentage of puncta-containing cells among at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also examined in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM. The results are expressed relative to the full control. (Scale bar: 5 µm.)

    Article Snippet: The polyclonal antibodies used in the IFAs were anti-LC3 (1:500; MBL International) and Alexa Fluor 647-conjugated anti-LC3 (1:250; Novus Biologicals).

    Techniques: Transfection

    ATG5 is involved in the IFN-γ–mediated elimination of liver-stage P. vivax . ( A–C ) HC04 cells were depleted of ATG5 by siRNA-mediated knockdown and cotransfected with cDNAs encoding RFP-GFP-LC3. At 48 h posttransfection, the cells were treated with IFN-γ for 4 h and then processed for fluorescence microscopy. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified by determining the percentage of puncta-containing cells among at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also analyzed in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM; the results are expressed relative to the full control. N.S., not significant. (Scale bar: 5 µm.) ( D ) Western blot analysis of ATG5 levels after knockdown of the protein. HC04 cells were transfected with scramble control siRNAs or siRNAs against ATG5. At 48 h posttransfection, the cells were harvested for immunoblot analysis. ( E and F ) ATG5-depleted or control HC04 cells infected with P. vivax sporozoites were treated with IFN-γ for 4 h, washed, and then maintained in complete medium until being harvested on day 4 for IFA. Data are mean ± SEM from at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: LAP-like process as an immune mechanism downstream of IFN-γ in control of the human malaria Plasmodium vivax liver stage

    doi: 10.1073/pnas.1525606113

    Figure Lengend Snippet: ATG5 is involved in the IFN-γ–mediated elimination of liver-stage P. vivax . ( A–C ) HC04 cells were depleted of ATG5 by siRNA-mediated knockdown and cotransfected with cDNAs encoding RFP-GFP-LC3. At 48 h posttransfection, the cells were treated with IFN-γ for 4 h and then processed for fluorescence microscopy. RFP + GFP + -LC3 (autophagosomes) and RFP + GFP − -LC3 (autolysosomes) were quantified by determining the percentage of puncta-containing cells among at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also analyzed in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM; the results are expressed relative to the full control. N.S., not significant. (Scale bar: 5 µm.) ( D ) Western blot analysis of ATG5 levels after knockdown of the protein. HC04 cells were transfected with scramble control siRNAs or siRNAs against ATG5. At 48 h posttransfection, the cells were harvested for immunoblot analysis. ( E and F ) ATG5-depleted or control HC04 cells infected with P. vivax sporozoites were treated with IFN-γ for 4 h, washed, and then maintained in complete medium until being harvested on day 4 for IFA. Data are mean ± SEM from at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.)

    Article Snippet: The polyclonal antibodies used in the IFAs were anti-LC3 (1:500; MBL International) and Alexa Fluor 647-conjugated anti-LC3 (1:250; Novus Biologicals).

    Techniques: Fluorescence, Microscopy, Western Blot, Transfection, Infection, Immunofluorescence

    Either PPARA or TFEB enhances phagosomal colocalization with autophagosomes/lysosomes, and antimicrobial responses against Mtb infection. (a-c and e-g) BMDMs from Sirt3 +/+ and sirt3 −/- mice were transduced with a control adenovirus, virus expressing a mouse PPARA (a-c) or TFEB (e-g) plasmid for 36 h. (a,b,e and f) The cells were infected with Mtb-ERFP (MOI = 10) for 4 h (a and e) or 6 h (b and f), and then stained with Alexa 488-conjugated LC3 Ab (green; for a or e), LAMP2 Ab (green; for b or f), and DAPI (blue; for nuclei). Representative immunofluorescence images of three independent replicates are shown. Scale bars: 5 µm. Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (c and g) The cells were infected with Mtb (MOI = 1) for 4 h, then lysed to determine intracellular bacterial loads at 3 dpi. G Right, qRT-PCR analysis for overexpression efficiency of adenovirus containing mouse TFEB plasmid or a control adenovirus. (d) Sirt3 +/+ and sirt3 −/- BMDMs were infected with Mtb (MOI = 10) at the indicated times, and then subjected to quantitative real-time PCR. * P

    Journal: Autophagy

    Article Title: SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions

    doi: 10.1080/15548627.2019.1582743

    Figure Lengend Snippet: Either PPARA or TFEB enhances phagosomal colocalization with autophagosomes/lysosomes, and antimicrobial responses against Mtb infection. (a-c and e-g) BMDMs from Sirt3 +/+ and sirt3 −/- mice were transduced with a control adenovirus, virus expressing a mouse PPARA (a-c) or TFEB (e-g) plasmid for 36 h. (a,b,e and f) The cells were infected with Mtb-ERFP (MOI = 10) for 4 h (a and e) or 6 h (b and f), and then stained with Alexa 488-conjugated LC3 Ab (green; for a or e), LAMP2 Ab (green; for b or f), and DAPI (blue; for nuclei). Representative immunofluorescence images of three independent replicates are shown. Scale bars: 5 µm. Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (c and g) The cells were infected with Mtb (MOI = 1) for 4 h, then lysed to determine intracellular bacterial loads at 3 dpi. G Right, qRT-PCR analysis for overexpression efficiency of adenovirus containing mouse TFEB plasmid or a control adenovirus. (d) Sirt3 +/+ and sirt3 −/- BMDMs were infected with Mtb (MOI = 10) at the indicated times, and then subjected to quantitative real-time PCR. * P

    Article Snippet: Cells were incubated with a primary anti-LC3 Ab (MBL International, PM036) or anti-LAMP2 Ab (Santa Cruz Biotechnology, sc-18822) at room temperature.

    Techniques: Infection, Mouse Assay, Transduction, Expressing, Plasmid Preparation, Staining, Immunofluorescence, Quantitative RT-PCR, Over Expression, Real-time Polymerase Chain Reaction

    SIRT3 activation enhances antibacterial autophagy, antimicrobial responses and ameliorates mitochondrial damage and oxidative stress during mycobacterial infection. (a-c) BMDMs from Sirt3 +/+ mice were stimulated with HKL (20 uM) for 24 h. Alexa Fluor 488-conjugated LC3 (green) and DAPI (blue) were detected by confocal microscopic analysis. (b) Quantitative analysis of LC3 puncta per cell. (c) Flow cytometric analysis of LC3B expression. Average MFIs of LC3B expression. (d) Sirt3 +/+ and sirt3 −/- BMDMs were transduced with retroviruses expressing a tandem-tagged mCherry-EGFP-LC3B and then infected with Mtb (MOI = 10) for 24 h. Cells were mCherry or EGFP expressing LC3B was detected by confocal microscopy. Scale bar: 5 μm. (e-j) Sirt3 +/+ and sirt3 −/- BMDMs were infected with Mtb (MOI = 1 for e or MOI = 10 for f-j) for 4 h and then treated with HKL (2, 10, and 20 μM) for 3 days (e) or 24 h (f-j, HKL 20 μM). (e) Intracellular survival of Mtb assessed by CFU assay. (f and g) Mtb-ERFP (red), Alexa Fluor 488-conjugated LAMP2 (green), and DAPI (blue) were detected by confocal microscopy. (f) Representative immunofluorescence images of three independent replicates are shown. Scale bar: 5 μm. (g) Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (h-i) MitoSOX FACS analysis (Representative images, for H; quantitative analysis, for I). (j) FACS analysis for MitoTracker Deep Red and MitoTracker Green. **P

    Journal: Autophagy

    Article Title: SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions

    doi: 10.1080/15548627.2019.1582743

    Figure Lengend Snippet: SIRT3 activation enhances antibacterial autophagy, antimicrobial responses and ameliorates mitochondrial damage and oxidative stress during mycobacterial infection. (a-c) BMDMs from Sirt3 +/+ mice were stimulated with HKL (20 uM) for 24 h. Alexa Fluor 488-conjugated LC3 (green) and DAPI (blue) were detected by confocal microscopic analysis. (b) Quantitative analysis of LC3 puncta per cell. (c) Flow cytometric analysis of LC3B expression. Average MFIs of LC3B expression. (d) Sirt3 +/+ and sirt3 −/- BMDMs were transduced with retroviruses expressing a tandem-tagged mCherry-EGFP-LC3B and then infected with Mtb (MOI = 10) for 24 h. Cells were mCherry or EGFP expressing LC3B was detected by confocal microscopy. Scale bar: 5 μm. (e-j) Sirt3 +/+ and sirt3 −/- BMDMs were infected with Mtb (MOI = 1 for e or MOI = 10 for f-j) for 4 h and then treated with HKL (2, 10, and 20 μM) for 3 days (e) or 24 h (f-j, HKL 20 μM). (e) Intracellular survival of Mtb assessed by CFU assay. (f and g) Mtb-ERFP (red), Alexa Fluor 488-conjugated LAMP2 (green), and DAPI (blue) were detected by confocal microscopy. (f) Representative immunofluorescence images of three independent replicates are shown. Scale bar: 5 μm. (g) Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (h-i) MitoSOX FACS analysis (Representative images, for H; quantitative analysis, for I). (j) FACS analysis for MitoTracker Deep Red and MitoTracker Green. **P

    Article Snippet: Cells were incubated with a primary anti-LC3 Ab (MBL International, PM036) or anti-LAMP2 Ab (Santa Cruz Biotechnology, sc-18822) at room temperature.

    Techniques: Activation Assay, Infection, Mouse Assay, Expressing, Transduction, Confocal Microscopy, Colony-forming Unit Assay, Immunofluorescence, FACS

    SIRT3 is essential for activation of antibacterial autophagy and phagosomal colocalization with lysosomes during mycobacterial infection. (a-e) BMDMs from Sirt3 +/+ and sirt3 −/- mice were infected with Mtb (a) or Mtb-ERFP (b-e) at MOI of 10, and incubated for 24 h (a), 4 h (b and c) or 6 h (d and e). (a) Representative images of FACS analysis for LC3B. Right, quantification of the results at left. (b-e) Cells were stained with Alexa 488-conjugated LC3 Ab (green; for b), LAMP2 Ab (green; for d), and DAPI (blue; for nuclei). (b and d) Representative immunofluorescence images of three independent replicates are shown. Scale bars: 5 µm. (c and e) Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (f) Sirt3 +/+ and sirt3 −/- mice were infected intranasally with Mtb (3 × 10 4 CFU), and monitored at 7 dpi. Below, the enlarged TEM images of the selected areas (asterisks) of Sirt3 +/+ and sirt3 −/- lung tissues. Representative TEM images from three independent experiments are shown. Scale bars: 5 µm. (g) Quantitation of 100 internalized mycobacteria per experimental condition. (H-J) BMDMs from Sirt3 +/+ and sirt3 −/- mice were transduced with a control adenovirus, virus expressing a mouse SIRT3 plasmid for 36 h. (h) The cells were infected with Mtb-ERFP (MOI = 10) for 4 h, and then stained with Alexa Fluor 488-conjugated LC3 Ab (green) and DAPI (blue; for nuclei). Representative immunofluorescence images of three independent replicates are shown. Scale bars: 5 µm. Right, qPCR analysis for overexpression efficiency of adenovirus containing mouse SIRT3 plasmid or a control adenovirus. (i) Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (j) The cells were infected with Mtb (MOI = 1) for 4 h, then lysed to determine intracellular bacterial loads at 3 dpi. *** P

    Journal: Autophagy

    Article Title: SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions

    doi: 10.1080/15548627.2019.1582743

    Figure Lengend Snippet: SIRT3 is essential for activation of antibacterial autophagy and phagosomal colocalization with lysosomes during mycobacterial infection. (a-e) BMDMs from Sirt3 +/+ and sirt3 −/- mice were infected with Mtb (a) or Mtb-ERFP (b-e) at MOI of 10, and incubated for 24 h (a), 4 h (b and c) or 6 h (d and e). (a) Representative images of FACS analysis for LC3B. Right, quantification of the results at left. (b-e) Cells were stained with Alexa 488-conjugated LC3 Ab (green; for b), LAMP2 Ab (green; for d), and DAPI (blue; for nuclei). (b and d) Representative immunofluorescence images of three independent replicates are shown. Scale bars: 5 µm. (c and e) Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (f) Sirt3 +/+ and sirt3 −/- mice were infected intranasally with Mtb (3 × 10 4 CFU), and monitored at 7 dpi. Below, the enlarged TEM images of the selected areas (asterisks) of Sirt3 +/+ and sirt3 −/- lung tissues. Representative TEM images from three independent experiments are shown. Scale bars: 5 µm. (g) Quantitation of 100 internalized mycobacteria per experimental condition. (H-J) BMDMs from Sirt3 +/+ and sirt3 −/- mice were transduced with a control adenovirus, virus expressing a mouse SIRT3 plasmid for 36 h. (h) The cells were infected with Mtb-ERFP (MOI = 10) for 4 h, and then stained with Alexa Fluor 488-conjugated LC3 Ab (green) and DAPI (blue; for nuclei). Representative immunofluorescence images of three independent replicates are shown. Scale bars: 5 µm. Right, qPCR analysis for overexpression efficiency of adenovirus containing mouse SIRT3 plasmid or a control adenovirus. (i) Quantitative data of colocalization analyses showing the means ± SEM of three independent experiments, with each experiment including at least 100 internalized mycobacteria scored in seven random fields. (j) The cells were infected with Mtb (MOI = 1) for 4 h, then lysed to determine intracellular bacterial loads at 3 dpi. *** P

    Article Snippet: Cells were incubated with a primary anti-LC3 Ab (MBL International, PM036) or anti-LAMP2 Ab (Santa Cruz Biotechnology, sc-18822) at room temperature.

    Techniques: Activation Assay, Infection, Mouse Assay, Incubation, FACS, Staining, Immunofluorescence, Transmission Electron Microscopy, Quantitation Assay, Transduction, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Over Expression

    LC3 activation is required for melanogenesis. Inhibition of LC3 attenuates melanin synthesis and tyrosinase activity in melanocytes. Melan-a cells were transfected with specific siRNAs for LC3, beclin-1, ATG5, or control siRNA for 24 h and were analyzed by Western blotting after autophagy induction (a,b) . Quantification of melanin (c) and tyrosinase activity (d) was performed for each transfected cell line. Results shown are the mean of three independent experiments ± SD. * P

    Journal: Scientific Reports

    Article Title: Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    doi: 10.1038/srep19914

    Figure Lengend Snippet: LC3 activation is required for melanogenesis. Inhibition of LC3 attenuates melanin synthesis and tyrosinase activity in melanocytes. Melan-a cells were transfected with specific siRNAs for LC3, beclin-1, ATG5, or control siRNA for 24 h and were analyzed by Western blotting after autophagy induction (a,b) . Quantification of melanin (c) and tyrosinase activity (d) was performed for each transfected cell line. Results shown are the mean of three independent experiments ± SD. * P

    Article Snippet: Primary antibodies included anti-LC3 (1:100 dilution; Santa Cruz Biotechnology), anti-MART1 (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA).

    Techniques: Activation Assay, Inhibition, Activity Assay, Transfection, Western Blot

    Autophagy induction promotes melanogenesis. (a,b) Melan-a melanocytes were treated with indicated concentration of rapamycin (Rap) or PP242 for 24 h and analyzed by Western blotting with antibodies against LC3, beclin-1, ATG5, p62, MITF, PMEL17, tyrosinase, and β-actin. Quantitative densitometry of protein expression compared to β-actin is shown. To determine the effect of autophagy activation on melanogenesis, cells were assayed for melanin content (c) and tyrosinase activity (d) . The effect of Rap and PP242 on cell proliferation (e) and viability (f) was determined at 24 h post-treatment. All results shown are the mean of three independent experiments ± SD. * P

    Journal: Scientific Reports

    Article Title: Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    doi: 10.1038/srep19914

    Figure Lengend Snippet: Autophagy induction promotes melanogenesis. (a,b) Melan-a melanocytes were treated with indicated concentration of rapamycin (Rap) or PP242 for 24 h and analyzed by Western blotting with antibodies against LC3, beclin-1, ATG5, p62, MITF, PMEL17, tyrosinase, and β-actin. Quantitative densitometry of protein expression compared to β-actin is shown. To determine the effect of autophagy activation on melanogenesis, cells were assayed for melanin content (c) and tyrosinase activity (d) . The effect of Rap and PP242 on cell proliferation (e) and viability (f) was determined at 24 h post-treatment. All results shown are the mean of three independent experiments ± SD. * P

    Article Snippet: Primary antibodies included anti-LC3 (1:100 dilution; Santa Cruz Biotechnology), anti-MART1 (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA).

    Techniques: Concentration Assay, Western Blot, Expressing, Activation Assay, Activity Assay

    LC3 modulates α-MSH-induced ERK and CREB activation, resulting in MITF expression. Knockdown of LC3 decreases α-MSH-induced melanogenesis. Melan-a cells transfected with control siRNA (siCon) or LC3 siRNA (siLC3) were treated with or without α-MSH (0.1 or 1 μM), and expression of LC3-I/II, pERK, ERK, MITF, and β-actin via Western blot and densitometry was measured (a,b) . The melanin index of treated versus control cells is also shown (c). Results shown are the mean of three independent experiments ± SD. * P

    Journal: Scientific Reports

    Article Title: Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    doi: 10.1038/srep19914

    Figure Lengend Snippet: LC3 modulates α-MSH-induced ERK and CREB activation, resulting in MITF expression. Knockdown of LC3 decreases α-MSH-induced melanogenesis. Melan-a cells transfected with control siRNA (siCon) or LC3 siRNA (siLC3) were treated with or without α-MSH (0.1 or 1 μM), and expression of LC3-I/II, pERK, ERK, MITF, and β-actin via Western blot and densitometry was measured (a,b) . The melanin index of treated versus control cells is also shown (c). Results shown are the mean of three independent experiments ± SD. * P

    Article Snippet: Primary antibodies included anti-LC3 (1:100 dilution; Santa Cruz Biotechnology), anti-MART1 (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA).

    Techniques: Activation Assay, Expressing, Transfection, Western Blot

    LC3 activation involves MITF expression during autophagy-induced melanogenesis. (a–f) Knockdown of LC3 decreases autophagy-induced melanogenesis. Melan-a cells were transfected with control siRNA (siCon) or LC3-specific siRNA (siLC3) and treated with (Rap) or without (None). Loss of Rap-induced melanosome formation following transfection with LC3 siRNA compared with control cells was observed by electron microscopy (a) and quantified ( b) . Scale bar, 5 μm. Data represent ± standard error of the mean (SEM) (* P

    Journal: Scientific Reports

    Article Title: Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    doi: 10.1038/srep19914

    Figure Lengend Snippet: LC3 activation involves MITF expression during autophagy-induced melanogenesis. (a–f) Knockdown of LC3 decreases autophagy-induced melanogenesis. Melan-a cells were transfected with control siRNA (siCon) or LC3-specific siRNA (siLC3) and treated with (Rap) or without (None). Loss of Rap-induced melanosome formation following transfection with LC3 siRNA compared with control cells was observed by electron microscopy (a) and quantified ( b) . Scale bar, 5 μm. Data represent ± standard error of the mean (SEM) (* P

    Article Snippet: Primary antibodies included anti-LC3 (1:100 dilution; Santa Cruz Biotechnology), anti-MART1 (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA).

    Techniques: Activation Assay, Expressing, Transfection, Electron Microscopy

    LC3 activation potentiates the melanogenic signals of α-MSH-sensitive melanoma cells. (a) Inhibition of LC3 suppresses α-MSH-induced melanogenesis. B16F10 melanoma cells transfected with control siRNA (siCon) or LC3 siRNA (siLC3) were treated with or without 0.1 μΜ α-MSH, after which their melanin content was measured. Results shown are the mean of three independent experiments ± SD. * P

    Journal: Scientific Reports

    Article Title: Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    doi: 10.1038/srep19914

    Figure Lengend Snippet: LC3 activation potentiates the melanogenic signals of α-MSH-sensitive melanoma cells. (a) Inhibition of LC3 suppresses α-MSH-induced melanogenesis. B16F10 melanoma cells transfected with control siRNA (siCon) or LC3 siRNA (siLC3) were treated with or without 0.1 μΜ α-MSH, after which their melanin content was measured. Results shown are the mean of three independent experiments ± SD. * P

    Article Snippet: Primary antibodies included anti-LC3 (1:100 dilution; Santa Cruz Biotechnology), anti-MART1 (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA).

    Techniques: Activation Assay, Inhibition, Transfection

    Autophagy activation correlates with melanogenesis in melanocytic nevi. (a) LC3 expression coincides with melanogenesis in sun-exposed skin of Korean patients. Immunohistochemical (top) or immunofluorescence staining (bottom) with anti-LC3 antibody (green) and anti-MART1 (melan A; red) antibody was performed to determine the extent to which LC3 expression is associated with melanin pigment in sun-exposed skin. The inset box represents an area shown at higher magnification (1000×). Magnification, 400×; Scale bar, 200 μm. (b–d) The relationship between autophagy flow and melanin synthesis in Melan-a melanocytes in the presence or absence of autophagy activator rapamycin (Rap) at indicated concentration for 24 h was determined by detecting conversion of LC3-I to LC3-II and p62 degradation using Western blot analysis. A subset of cells was also exposed to the lysosomal inhibitor bafilomycin A1 (Baf.A). Protein expression was determined via quantitative densitometry of experimental proteins compared to β-actin expression (b,c) , and melanogenesis was estimated by measuring melanin content (d) compared to control cells. * P

    Journal: Scientific Reports

    Article Title: Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    doi: 10.1038/srep19914

    Figure Lengend Snippet: Autophagy activation correlates with melanogenesis in melanocytic nevi. (a) LC3 expression coincides with melanogenesis in sun-exposed skin of Korean patients. Immunohistochemical (top) or immunofluorescence staining (bottom) with anti-LC3 antibody (green) and anti-MART1 (melan A; red) antibody was performed to determine the extent to which LC3 expression is associated with melanin pigment in sun-exposed skin. The inset box represents an area shown at higher magnification (1000×). Magnification, 400×; Scale bar, 200 μm. (b–d) The relationship between autophagy flow and melanin synthesis in Melan-a melanocytes in the presence or absence of autophagy activator rapamycin (Rap) at indicated concentration for 24 h was determined by detecting conversion of LC3-I to LC3-II and p62 degradation using Western blot analysis. A subset of cells was also exposed to the lysosomal inhibitor bafilomycin A1 (Baf.A). Protein expression was determined via quantitative densitometry of experimental proteins compared to β-actin expression (b,c) , and melanogenesis was estimated by measuring melanin content (d) compared to control cells. * P

    Article Snippet: Primary antibodies included anti-LC3 (1:100 dilution; Santa Cruz Biotechnology), anti-MART1 (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA).

    Techniques: Activation Assay, Expressing, Immunohistochemistry, Immunofluorescence, Staining, Flow Cytometry, Concentration Assay, Western Blot

    Proposed model of LC3’s role in melanogenesis during stress response. This schematic describes our proposed model of the signaling pathways involved in melanogenesis and their potential link to autophagy activation. Black arrows depict steps that have been experimentally verified from previous reports, whereas dashed arrows indicate unconfirmed steps. Red arrows represent experimental results from our current study: ( i ) Autophagy activation increases LC3-II levels, which triggers ERK and CREB phosphorylation and ( ii ) ERK-CREB activation leads to increased MITF expression and subsequent melanogenesis. The results reported here suggest that LC3 is a mediator that potentiates melanogenic signaling via α-MSH and MITF under certain stress conditions (e.g. autophagy activation) in melanocytes.

    Journal: Scientific Reports

    Article Title: Microtubule-associated protein light chain 3 is involved in melanogenesis via regulation of MITF expression in melanocytes

    doi: 10.1038/srep19914

    Figure Lengend Snippet: Proposed model of LC3’s role in melanogenesis during stress response. This schematic describes our proposed model of the signaling pathways involved in melanogenesis and their potential link to autophagy activation. Black arrows depict steps that have been experimentally verified from previous reports, whereas dashed arrows indicate unconfirmed steps. Red arrows represent experimental results from our current study: ( i ) Autophagy activation increases LC3-II levels, which triggers ERK and CREB phosphorylation and ( ii ) ERK-CREB activation leads to increased MITF expression and subsequent melanogenesis. The results reported here suggest that LC3 is a mediator that potentiates melanogenic signaling via α-MSH and MITF under certain stress conditions (e.g. autophagy activation) in melanocytes.

    Article Snippet: Primary antibodies included anti-LC3 (1:100 dilution; Santa Cruz Biotechnology), anti-MART1 (1:300 dilution; Medical & Biological Laboratories, Nagoya, Japan) and mouse IgG1/kappa (1:50 dilution; eBioscience, Inc., San Diego, CA, USA).

    Techniques: Activation Assay, Expressing

    In vivo accumulation of LC3 and LAMP-1 around T. gondii in brain endothelial cells and effects of administration of an inhibitor of autophagy on hematogenous invasion of the brain and eye by T. gondii . ( a ) Mice received T. gondii -infected dendritic cells i.v. After 18 h, mice were perfused and brain sections were stained with Tomato lectin-DyLight 488, anti- T. gondii Ab plus Alexa 568-conjugated secondary Ab and anti-LC3 or anti-LAMP-1 Abs plus Alexa 647-conjugated secondary Abs. Images to the left show parasites present in Tomato lectin + cells (endothelial cells). The images from the Trg-Ctr mouse show a parasite not surrounded by accumulation of LC3 or LAMP-1. Ring-like accumulation of LC3 or LAMP-1 around parasite (arrowheads) is noted in images from the Trg-DN EGFR mouse (X630). Bar, 5 μm. Bar graphs represent percentages of parasites present in endothelial cells that were surrounded by accumulation of LC3 or LAMP-1 (mean ± SEM from 2 pooled experiments). ** p

    Journal: Scientific Reports

    Article Title: Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii

    doi: 10.1038/s41598-018-36724-2

    Figure Lengend Snippet: In vivo accumulation of LC3 and LAMP-1 around T. gondii in brain endothelial cells and effects of administration of an inhibitor of autophagy on hematogenous invasion of the brain and eye by T. gondii . ( a ) Mice received T. gondii -infected dendritic cells i.v. After 18 h, mice were perfused and brain sections were stained with Tomato lectin-DyLight 488, anti- T. gondii Ab plus Alexa 568-conjugated secondary Ab and anti-LC3 or anti-LAMP-1 Abs plus Alexa 647-conjugated secondary Abs. Images to the left show parasites present in Tomato lectin + cells (endothelial cells). The images from the Trg-Ctr mouse show a parasite not surrounded by accumulation of LC3 or LAMP-1. Ring-like accumulation of LC3 or LAMP-1 around parasite (arrowheads) is noted in images from the Trg-DN EGFR mouse (X630). Bar, 5 μm. Bar graphs represent percentages of parasites present in endothelial cells that were surrounded by accumulation of LC3 or LAMP-1 (mean ± SEM from 2 pooled experiments). ** p

    Article Snippet: Brain sections were also stained with anti-LC3 (Abgent) or anti-LAMP-1 (Developmental Studies Hybridoma Bank) Abs.

    Techniques: In Vivo, Mouse Assay, Infection, Staining

    Brain endothelial cell from Trg-DN EGFR exhibit spontaneous targeting of intracellular T. gondii by LC3 and LAMP-1 as well as killing of the parasite dependent on autophagy. ( a ) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RH T. gondii . Monolayers were assessed by light microscopy. The percentages of infected cells, numbers of vacuoles containing T. gondii per 100 endothelial cells and tachyzoites per 100 endothelial cells were determined at 2 and 24 h. ( b ) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RH T. gondii . Lysates obtained at 0, 5 and 15 min post-challenge were subjected to immunoblot as indicated. ( c , d ) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RFP T. gondii (RH). Expression of LC3 was assessed by immunofluorescence 5 h after challenge ( c ). Insets represent magnification of areas around the parasites. Arrowheads indicate LC3 accumulation around the parasite (X630). Bar, 5 μm. ( d ) Expression of LAMP-1 was assessed by immunofluorescence 8 h after challenge. Arrowheads indicate LAMP-1 accumulation around the parasite. Bar, 5 μm. Bar graphs represent percentages of parasitophorous vacuoles surrounded by ring-like accumulation of LC3 or LAMP-1. ( e ) Endothelial cells were challenged with RH T. gondii followed by addition of lysosomal inhibitors (LI; leupeptin and pepstatin). Cells were examined as above. ( f ) Endothelial cells were transfected with control or ULK1 siRNA followed by challenge with RH T. gondii . Bars are mean ± SEM of 6 samples per group pooled from 2–3 experiments. ** p

    Journal: Scientific Reports

    Article Title: Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii

    doi: 10.1038/s41598-018-36724-2

    Figure Lengend Snippet: Brain endothelial cell from Trg-DN EGFR exhibit spontaneous targeting of intracellular T. gondii by LC3 and LAMP-1 as well as killing of the parasite dependent on autophagy. ( a ) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RH T. gondii . Monolayers were assessed by light microscopy. The percentages of infected cells, numbers of vacuoles containing T. gondii per 100 endothelial cells and tachyzoites per 100 endothelial cells were determined at 2 and 24 h. ( b ) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RH T. gondii . Lysates obtained at 0, 5 and 15 min post-challenge were subjected to immunoblot as indicated. ( c , d ) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RFP T. gondii (RH). Expression of LC3 was assessed by immunofluorescence 5 h after challenge ( c ). Insets represent magnification of areas around the parasites. Arrowheads indicate LC3 accumulation around the parasite (X630). Bar, 5 μm. ( d ) Expression of LAMP-1 was assessed by immunofluorescence 8 h after challenge. Arrowheads indicate LAMP-1 accumulation around the parasite. Bar, 5 μm. Bar graphs represent percentages of parasitophorous vacuoles surrounded by ring-like accumulation of LC3 or LAMP-1. ( e ) Endothelial cells were challenged with RH T. gondii followed by addition of lysosomal inhibitors (LI; leupeptin and pepstatin). Cells were examined as above. ( f ) Endothelial cells were transfected with control or ULK1 siRNA followed by challenge with RH T. gondii . Bars are mean ± SEM of 6 samples per group pooled from 2–3 experiments. ** p

    Article Snippet: Brain sections were also stained with anti-LC3 (Abgent) or anti-LAMP-1 (Developmental Studies Hybridoma Bank) Abs.

    Techniques: Mouse Assay, Infection, Light Microscopy, Expressing, Immunofluorescence, Transfection