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    Vector Laboratories bsa
    Bsa, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 2792 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cleaved caspase 3
    miR-132 supplementation reduces Tau pathology and neuronal loss in PS19 mice. a Representative IHC images of CA1 and adjacent cortical layers stained for NeuN, cleaved <t>Caspase-3,</t> PHF-Tau, glial fibrillary acidic protein (GFAP), and DAPI staining. The cells positive for the activated caspase-3 and PHF-Tau are marked by arrows. Scale bar = 100 µm. b Tangle-like PHF-positive inclusions (in green) observed in the cell bodies of untreated/EV-treated neurons, and the less intense and more evenly distributed PHF staining in the soma and neurites of apparently intact neurons from miR-132-treated mice. Scale bar = 100 µm. c – f Image J quantification of the marker-positive cells per field in the CA1 area. For all images and quantifications, n = 14 mice per condition, 15 sections per brain. All graphical data are shown as mean ± SEM, Student’s t test, * P
    Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 43756 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies immunohistochemical staining
    WT1 <t>immunostaining</t> in fetal adrenal gland. WT1 immunoreactivity was located in the cytoplasm of cells of the fetal zone and in the adrenal capsule, with some strongly reactive capsular cells intermingled with non-reactive cells (F, fetal zone; D, definitive zone; C, adrenal capsule).
    Immunohistochemical Staining, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 2699 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt
    Antrocin inhibits the <t>PI3K/AKT</t> and MAPK signaling pathways and phosphorylation of IGF-1R. PC3-KD cells were treated with IR (2 Gy), antrocin (100 μM), or IR plus antrocin, followed by incubation for 48 h. ( A ) The represented gene network was identified by ingenuity pathway analysis. The color of each line indicates the regulation of gene expression. ( B ) Cell lysates were harvested and subjected to Western blot analysis using antibodies against p85, p110, p-AKT, AKT, <t>GSK3-β,</t> p-GSK3-β, p-β-catenin, and β-catenin, and ( C ) antibodies against IGF-1R, p38, JNK, Erk1/2, and their respective phosphorylated forms. The data represent one of three independent experiments. β-actin was used as a loading control. The expression level of each protein was quantified by the signal intensity and normalized with each vehicle untreated group. The relative level of each protein expression was indicated at the bottom of each lane.
    Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 24236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ihc  (Abcam)
    94
    Abcam ihc
    Antrocin inhibits the <t>PI3K/AKT</t> and MAPK signaling pathways and phosphorylation of IGF-1R. PC3-KD cells were treated with IR (2 Gy), antrocin (100 μM), or IR plus antrocin, followed by incubation for 48 h. ( A ) The represented gene network was identified by ingenuity pathway analysis. The color of each line indicates the regulation of gene expression. ( B ) Cell lysates were harvested and subjected to Western blot analysis using antibodies against p85, p110, p-AKT, AKT, <t>GSK3-β,</t> p-GSK3-β, p-β-catenin, and β-catenin, and ( C ) antibodies against IGF-1R, p38, JNK, Erk1/2, and their respective phosphorylated forms. The data represent one of three independent experiments. β-actin was used as a loading control. The expression level of each protein was quantified by the signal intensity and normalized with each vehicle untreated group. The relative level of each protein expression was indicated at the bottom of each lane.
    Ihc, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho akt
    Effects of melatonin on the activation of the <t>PI3K/AKT</t> and MAPK- <t>ERK1/2</t> pathways(A). Effect of inhibition of LY294002 and wortmannin on the activation of the PI3K/AKT pathway (B, C). Effect of MTR inhibitors on the activation of the PI3K/AKT pathway (D) in the TI in small follicles. Small follicles were treated with 10 ng/mL melatonin for 0 min, 30 min, 1 h, or 4 h with or without pretreatment with a combination of inhibitors, 25 μM LY294002, 0.1 μM wortmannin, 10 μM luzindole, or 10 μM 4P-PDOT for 30 min. TI lysates were subjected to SDS-PAGE/immunoblotting for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 analysis. The results are the mean ± SEM of three independent experiments. The relative density ratio was calculated on the basis of a control group value of one. * P
    Anti Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ventana Medical immunostaining
    Effects of melatonin on the activation of the <t>PI3K/AKT</t> and MAPK- <t>ERK1/2</t> pathways(A). Effect of inhibition of LY294002 and wortmannin on the activation of the PI3K/AKT pathway (B, C). Effect of MTR inhibitors on the activation of the PI3K/AKT pathway (D) in the TI in small follicles. Small follicles were treated with 10 ng/mL melatonin for 0 min, 30 min, 1 h, or 4 h with or without pretreatment with a combination of inhibitors, 25 μM LY294002, 0.1 μM wortmannin, 10 μM luzindole, or 10 μM 4P-PDOT for 30 min. TI lysates were subjected to SDS-PAGE/immunoblotting for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 analysis. The results are the mean ± SEM of three independent experiments. The relative density ratio was calculated on the basis of a control group value of one. * P
    Immunostaining, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 92/100, based on 413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ventana Medical immunostainer
    Effects of melatonin on the activation of the <t>PI3K/AKT</t> and MAPK- <t>ERK1/2</t> pathways(A). Effect of inhibition of LY294002 and wortmannin on the activation of the PI3K/AKT pathway (B, C). Effect of MTR inhibitors on the activation of the PI3K/AKT pathway (D) in the TI in small follicles. Small follicles were treated with 10 ng/mL melatonin for 0 min, 30 min, 1 h, or 4 h with or without pretreatment with a combination of inhibitors, 25 μM LY294002, 0.1 μM wortmannin, 10 μM luzindole, or 10 μM 4P-PDOT for 30 min. TI lysates were subjected to SDS-PAGE/immunoblotting for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 analysis. The results are the mean ± SEM of three independent experiments. The relative density ratio was calculated on the basis of a control group value of one. * P
    Immunostainer, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 92/100, based on 2121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam ihc analysis
    Effects of melatonin on the activation of the <t>PI3K/AKT</t> and MAPK- <t>ERK1/2</t> pathways(A). Effect of inhibition of LY294002 and wortmannin on the activation of the PI3K/AKT pathway (B, C). Effect of MTR inhibitors on the activation of the PI3K/AKT pathway (D) in the TI in small follicles. Small follicles were treated with 10 ng/mL melatonin for 0 min, 30 min, 1 h, or 4 h with or without pretreatment with a combination of inhibitors, 25 μM LY294002, 0.1 μM wortmannin, 10 μM luzindole, or 10 μM 4P-PDOT for 30 min. TI lysates were subjected to SDS-PAGE/immunoblotting for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 analysis. The results are the mean ± SEM of three independent experiments. The relative density ratio was calculated on the basis of a control group value of one. * P
    Ihc Analysis, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies immunohistochemistry immunohistochemical staining
    Ghrelin expression in node negative breast cancer tissue was analyzed by <t>immunohistochemistry.</t> Representative images of ghrelin with 0 (non-immunoreactive), 1 (weak), 2 (moderate) and 3 (strong) <t>immunostaining.</t> Scale bar = 100 μm.
    Immunohistochemistry Immunohistochemical Staining, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc pd l1
    Etoposide inhibits the <t>EMT/β-catenin/STT3/PD-L1</t> axis through TOP2B degradation-dependent nuclear β-catenin downregulation. a Effect of TOP2 isoforms knockdown on the expression of EMT markers, STT3 and PD-L1 in 4T1 cell. b Representative phase-contrast microscopy images of 4T1 cell treated with indicated sg-RNAs. Scale bar, 50 μm. c Influence of TOP2 isoforms knockdown on etoposide-induced MET and downregulation of STT3 and PD-L1. d Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated cancer cell killing assay (bottom right) of tumorspheres cultured from various sgRNA-treated 4T1 cells. e Effect of proteasome inhibitor (MG132) and caspase inhibitor (Z-VAD-FMK) on etoposide-induced TOP2B degradation, MET, and downregulation of STT3 and PD-L1. f Western blotting of whole-cell extracts analyzing the effect of etoposide and TOP2B knockdown on total and active (non-phospho) β-catenin (β-Cat). g Western blotting of whole-cell extracts (WCE), cytosolic (Cyto), membrane (Mem), or nuclear (Nuc) fractions from sgRNA-treated 4T1 cells analyzing the effect of TOP2B knockdown on β-catenin subcellular localizations. Histone H3, E-cadherin (E-Cad), and tubulin were used as makers of nuclear, membrane, and cytosolic fractions, respectively. h Efficacy of β-catenin knockdown (si-β-Cat) in desensitizing cells to etoposide- and sg-TOP2B- induced MET and downregulation of STT3 and PD-L1. Nuc nuclear fraction, WCE whole-cell extracts. i Co-immunoprecipitation assay showing interaction between TOP2B and β-catenin. j Duolink assay analyzing the interaction of TOP2B and active (non-phospho) β-catenin in the nucleus. Red dots (indicated by arrows) represented TOP2B-β-catenin interaction signals and nuclei were counterstained with DAPI (blue). The right column shows higher-magnification image of the area outlined in the left column. Scale bar, 5 μm. Error bars represent s.d. ( n = 3). * P
    Pd L1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher immunostaining
    Etoposide inhibits the <t>EMT/β-catenin/STT3/PD-L1</t> axis through TOP2B degradation-dependent nuclear β-catenin downregulation. a Effect of TOP2 isoforms knockdown on the expression of EMT markers, STT3 and PD-L1 in 4T1 cell. b Representative phase-contrast microscopy images of 4T1 cell treated with indicated sg-RNAs. Scale bar, 50 μm. c Influence of TOP2 isoforms knockdown on etoposide-induced MET and downregulation of STT3 and PD-L1. d Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated cancer cell killing assay (bottom right) of tumorspheres cultured from various sgRNA-treated 4T1 cells. e Effect of proteasome inhibitor (MG132) and caspase inhibitor (Z-VAD-FMK) on etoposide-induced TOP2B degradation, MET, and downregulation of STT3 and PD-L1. f Western blotting of whole-cell extracts analyzing the effect of etoposide and TOP2B knockdown on total and active (non-phospho) β-catenin (β-Cat). g Western blotting of whole-cell extracts (WCE), cytosolic (Cyto), membrane (Mem), or nuclear (Nuc) fractions from sgRNA-treated 4T1 cells analyzing the effect of TOP2B knockdown on β-catenin subcellular localizations. Histone H3, E-cadherin (E-Cad), and tubulin were used as makers of nuclear, membrane, and cytosolic fractions, respectively. h Efficacy of β-catenin knockdown (si-β-Cat) in desensitizing cells to etoposide- and sg-TOP2B- induced MET and downregulation of STT3 and PD-L1. Nuc nuclear fraction, WCE whole-cell extracts. i Co-immunoprecipitation assay showing interaction between TOP2B and β-catenin. j Duolink assay analyzing the interaction of TOP2B and active (non-phospho) β-catenin in the nucleus. Red dots (indicated by arrows) represented TOP2B-β-catenin interaction signals and nuclei were counterstained with DAPI (blue). The right column shows higher-magnification image of the area outlined in the left column. Scale bar, 5 μm. Error bars represent s.d. ( n = 3). * P
    Immunostaining, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2747 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad immunohistochemical staining
    Etoposide inhibits the <t>EMT/β-catenin/STT3/PD-L1</t> axis through TOP2B degradation-dependent nuclear β-catenin downregulation. a Effect of TOP2 isoforms knockdown on the expression of EMT markers, STT3 and PD-L1 in 4T1 cell. b Representative phase-contrast microscopy images of 4T1 cell treated with indicated sg-RNAs. Scale bar, 50 μm. c Influence of TOP2 isoforms knockdown on etoposide-induced MET and downregulation of STT3 and PD-L1. d Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated cancer cell killing assay (bottom right) of tumorspheres cultured from various sgRNA-treated 4T1 cells. e Effect of proteasome inhibitor (MG132) and caspase inhibitor (Z-VAD-FMK) on etoposide-induced TOP2B degradation, MET, and downregulation of STT3 and PD-L1. f Western blotting of whole-cell extracts analyzing the effect of etoposide and TOP2B knockdown on total and active (non-phospho) β-catenin (β-Cat). g Western blotting of whole-cell extracts (WCE), cytosolic (Cyto), membrane (Mem), or nuclear (Nuc) fractions from sgRNA-treated 4T1 cells analyzing the effect of TOP2B knockdown on β-catenin subcellular localizations. Histone H3, E-cadherin (E-Cad), and tubulin were used as makers of nuclear, membrane, and cytosolic fractions, respectively. h Efficacy of β-catenin knockdown (si-β-Cat) in desensitizing cells to etoposide- and sg-TOP2B- induced MET and downregulation of STT3 and PD-L1. Nuc nuclear fraction, WCE whole-cell extracts. i Co-immunoprecipitation assay showing interaction between TOP2B and β-catenin. j Duolink assay analyzing the interaction of TOP2B and active (non-phospho) β-catenin in the nucleus. Red dots (indicated by arrows) represented TOP2B-β-catenin interaction signals and nuclei were counterstained with DAPI (blue). The right column shows higher-magnification image of the area outlined in the left column. Scale bar, 5 μm. Error bars represent s.d. ( n = 3). * P
    Immunohistochemical Staining, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    APO BRDU IHC IMMUNOHISTOCHEMISTRY KIT APO BRDU IHCTM IMMUNOHISTOCHEMISTRY KIT is a two color TUNEL Terminal deoxynucleotide transferase dUTP Nick End Labelling immunohistochemical assay kit which enables the identification of
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    Image Search Results


    miR-132 supplementation reduces Tau pathology and neuronal loss in PS19 mice. a Representative IHC images of CA1 and adjacent cortical layers stained for NeuN, cleaved Caspase-3, PHF-Tau, glial fibrillary acidic protein (GFAP), and DAPI staining. The cells positive for the activated caspase-3 and PHF-Tau are marked by arrows. Scale bar = 100 µm. b Tangle-like PHF-positive inclusions (in green) observed in the cell bodies of untreated/EV-treated neurons, and the less intense and more evenly distributed PHF staining in the soma and neurites of apparently intact neurons from miR-132-treated mice. Scale bar = 100 µm. c – f Image J quantification of the marker-positive cells per field in the CA1 area. For all images and quantifications, n = 14 mice per condition, 15 sections per brain. All graphical data are shown as mean ± SEM, Student’s t test, * P

    Journal: Acta Neuropathologica

    Article Title: MicroRNA-132 provides neuroprotection for tauopathies via multiple signaling pathways

    doi: 10.1007/s00401-018-1880-5

    Figure Lengend Snippet: miR-132 supplementation reduces Tau pathology and neuronal loss in PS19 mice. a Representative IHC images of CA1 and adjacent cortical layers stained for NeuN, cleaved Caspase-3, PHF-Tau, glial fibrillary acidic protein (GFAP), and DAPI staining. The cells positive for the activated caspase-3 and PHF-Tau are marked by arrows. Scale bar = 100 µm. b Tangle-like PHF-positive inclusions (in green) observed in the cell bodies of untreated/EV-treated neurons, and the less intense and more evenly distributed PHF staining in the soma and neurites of apparently intact neurons from miR-132-treated mice. Scale bar = 100 µm. c – f Image J quantification of the marker-positive cells per field in the CA1 area. For all images and quantifications, n = 14 mice per condition, 15 sections per brain. All graphical data are shown as mean ± SEM, Student’s t test, * P

    Article Snippet: The 16-μm-thick sections were immunostained for NeuN, GFAP, Cleaved Caspase-3, and Tau-PFH with antibodies from Cell Signaling.

    Techniques: Mouse Assay, Immunohistochemistry, Staining, Marker

    CIRBP over-expression suppressed HIF-1α up-regulation in hypoxia and inhibited hypoxia-induced neuron apoptosis. Western blot analysis of CIRBP expression levels (A) and HIF-1α (B) in SH-SY5Y cells transfected with mock vector (pEGFP-N2) or pEGFP-N2-CIRBP (pEGFP-N2-C) cultured in normoxia or 1% hypoxia for 48 h. ** depicts the significant increase of CIRBP and decrease of HIF-1α transfected with pEGFP-N2-C in hypoxia group compared with control group. (C) Flow cytometry shows apoptosis of SH-SY5Y cells transfected with mock vector (pEGFP-N2) or pEGFP-N2-CIRBP (pEGFP-N2-C) cells by Annexin V and PI staining in control or hypoxia group for 48 hours. Bar graphs demonstrating extent of apoptotic cells, ** depicts the significant decrease of ratio of apoptotic cells in cells transfected with pEGFP-N2-CIRBP in hypoxia group compared with cells in control group. (D) TUNEL apoptosis detection of SH-SY5Y neuron-like cells transfected with pEGFP-N2 or pEGFP-N2-CIRBP cultured under normoxia or hypoxia for 48 hours. Decrease TUNEL-positive cells were observed in cells transfected with pEGFP-N2-CIRBP in hypoxia group compared with control groups. Bars = 100 μm. (E) Western blot analysis of caspase-3, cleaved caspase-3, Bcl-2, Bax and β-actin in SH-SY5Y neuron-like cells transfected with pEGFP-N2 or pEGFP-N2-CIRBP in control group or hypoxia. Graphs show relative ratio of cleaved caspase-3/caspase-3 and Bax/Bcl-2. Notice the significant decrease in cleaved caspase-3 and Bax/Bcl-2 ratio in cells transfected with pEGFP-N2-CIRBP in the hypoxia groups compared with the control group, respectively. ** (p

    Journal: International Journal of Biological Sciences

    Article Title: Cold Inducible RNA Binding Protein Is Involved in Chronic Hypoxia Induced Neuron Apoptosis by Down-Regulating HIF-1α Expression and Regulated By microRNA-23a

    doi: 10.7150/ijbs.17800

    Figure Lengend Snippet: CIRBP over-expression suppressed HIF-1α up-regulation in hypoxia and inhibited hypoxia-induced neuron apoptosis. Western blot analysis of CIRBP expression levels (A) and HIF-1α (B) in SH-SY5Y cells transfected with mock vector (pEGFP-N2) or pEGFP-N2-CIRBP (pEGFP-N2-C) cultured in normoxia or 1% hypoxia for 48 h. ** depicts the significant increase of CIRBP and decrease of HIF-1α transfected with pEGFP-N2-C in hypoxia group compared with control group. (C) Flow cytometry shows apoptosis of SH-SY5Y cells transfected with mock vector (pEGFP-N2) or pEGFP-N2-CIRBP (pEGFP-N2-C) cells by Annexin V and PI staining in control or hypoxia group for 48 hours. Bar graphs demonstrating extent of apoptotic cells, ** depicts the significant decrease of ratio of apoptotic cells in cells transfected with pEGFP-N2-CIRBP in hypoxia group compared with cells in control group. (D) TUNEL apoptosis detection of SH-SY5Y neuron-like cells transfected with pEGFP-N2 or pEGFP-N2-CIRBP cultured under normoxia or hypoxia for 48 hours. Decrease TUNEL-positive cells were observed in cells transfected with pEGFP-N2-CIRBP in hypoxia group compared with control groups. Bars = 100 μm. (E) Western blot analysis of caspase-3, cleaved caspase-3, Bcl-2, Bax and β-actin in SH-SY5Y neuron-like cells transfected with pEGFP-N2 or pEGFP-N2-CIRBP in control group or hypoxia. Graphs show relative ratio of cleaved caspase-3/caspase-3 and Bax/Bcl-2. Notice the significant decrease in cleaved caspase-3 and Bax/Bcl-2 ratio in cells transfected with pEGFP-N2-CIRBP in the hypoxia groups compared with the control group, respectively. ** (p

    Article Snippet: Rabbit anti-HIF-1α (Cat. number: 14179), rabbit anti-Bax (Cat. number: 14796), mouse anti-Bcl-2 (Cat. number: 15071), anti-caspase-3 (Cat. number: 9662) and anti-cleaved caspase-3 (Cat. number: 9664) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, Cell Culture, Flow Cytometry, Cytometry, Staining, TUNEL Assay

    Extensive cell death in layer 2-6 pyramidal cells. a , Coronal sections through the S1 cortex of P4 Nex Cre/+ ;Fucci2 (left) and P7 Nkx2-1-Cre;RCL tdTomato (right) mice immunostained for cleaved caspase-3 (yellow) and mCherry (green, left) or tdTomato (magenta, right). b , Quantification of density of cleaved caspase-3 cells in pyramidal neurons (left, green bars) and MGE interneurons (right, magenta bars) during postnatal development (for pyramidal neurons, ANOVA, F = 73.6, *** p = 0.003 [P2 vs P4], *** p = 0.00006 [P4 vs P7], n = 3 animals for all ages; for MGE interneurons, ANOVA, F = 16.91, * p = 0.027 [P5 vs P7], ** p = 0.0029 [P7 vs P10], n = 3 animals for all ages). c , Coronal sections through the barrel cortex of Nex Cre/+ ;Fucci2 mice during postnatal development immunostained for mCherry (green) and CTGF (yellow). d , Total number of pyramidal cells excluding subplate cells in the neocortex of Nex Cre/+ ;Fucci2 mice (ANOVA, F = 4.83 and * p = 0.03; n = 3 animals for P2 and P5, and 4 animals for P3, P4 and P21). e , Temporal variation in the percentage of pyramidal cells excluding the subplate contribution during postnatal development. Data is shown as mean ± SEM. Scale bars, 100 μm.

    Journal: Nature

    Article Title: Pyramidal cell regulation of interneuron survival sculpts cortical networks

    doi: 10.1038/s41586-018-0139-6

    Figure Lengend Snippet: Extensive cell death in layer 2-6 pyramidal cells. a , Coronal sections through the S1 cortex of P4 Nex Cre/+ ;Fucci2 (left) and P7 Nkx2-1-Cre;RCL tdTomato (right) mice immunostained for cleaved caspase-3 (yellow) and mCherry (green, left) or tdTomato (magenta, right). b , Quantification of density of cleaved caspase-3 cells in pyramidal neurons (left, green bars) and MGE interneurons (right, magenta bars) during postnatal development (for pyramidal neurons, ANOVA, F = 73.6, *** p = 0.003 [P2 vs P4], *** p = 0.00006 [P4 vs P7], n = 3 animals for all ages; for MGE interneurons, ANOVA, F = 16.91, * p = 0.027 [P5 vs P7], ** p = 0.0029 [P7 vs P10], n = 3 animals for all ages). c , Coronal sections through the barrel cortex of Nex Cre/+ ;Fucci2 mice during postnatal development immunostained for mCherry (green) and CTGF (yellow). d , Total number of pyramidal cells excluding subplate cells in the neocortex of Nex Cre/+ ;Fucci2 mice (ANOVA, F = 4.83 and * p = 0.03; n = 3 animals for P2 and P5, and 4 animals for P3, P4 and P21). e , Temporal variation in the percentage of pyramidal cells excluding the subplate contribution during postnatal development. Data is shown as mean ± SEM. Scale bars, 100 μm.

    Article Snippet: The following antibodies were used: goat anti-CTGF (1:200, Santa Cruz), rabbit anti cleaved-caspase-3 (1:200, Cell Signalling), rabbit anti-dsRed (1:500, Clontech), goat anti-mCherry (1:500, Antibodies-online), rabbit anti-GABA (1:2000, Millipore), mouse anti-GABA (1:500, Sigma), mouse anti-NeuN (1:500, Millipore) mouse anti-parvalbumin (1:1000, Swant), rabbit anti-parvalbumin (1:5000, Swant), rat anti-somatostatin (1:300, Millipore) and rabbit anti-PTEN (1:500, Abcam).

    Techniques: Mouse Assay

    CNO control experiments. a , Schematic of experimental design. b , Coronal sections through S1 of P8 Nex Cre mice injected with AAV8-dio-hM4Di-mCherry at P0 and treated with (+CNO) or without (-CNO) between P5 and P8, immunostained for cleaved caspase-3 (magenta) and counterstained with DAPI (grey). c , Quantification of the density of cleaved caspase-3 cells in P8 mice injected with hM4Di-mCherry and treated (magenta bar) or not treated (grey bar) with CNO between P5-P8 (2-tailed Student’s unpaired t -test, *** p =0.009, n = 8 animals for -CNO, and n = 7 animals for +CNO). d , Schematic of experimental design for CNO control experiments. e , Quantification of the density of PV (left) and SST (right) cells in P21 mice injected with hM3Dq-mCherry or hM4Di-mCherry and not treated with CNO (grey bars), or not injected with viruses and treated with CNO (magenta bars) between P5-P8 (ANOVA, p = 0.24 [PV] and p = 0.65 [SST] for PV, n = 7 animals for hM3Dq and hM4DI -CNO, 4 animals for non-injected +CNO; for SST, n = 9 animals for hM3Dq -CNO, 7 animals for hM4Di -CNO, and 4 animals for non-injected +CNO). Data is shown as mean ± SEM. Scale bar, 100 µm.

    Journal: Nature

    Article Title: Pyramidal cell regulation of interneuron survival sculpts cortical networks

    doi: 10.1038/s41586-018-0139-6

    Figure Lengend Snippet: CNO control experiments. a , Schematic of experimental design. b , Coronal sections through S1 of P8 Nex Cre mice injected with AAV8-dio-hM4Di-mCherry at P0 and treated with (+CNO) or without (-CNO) between P5 and P8, immunostained for cleaved caspase-3 (magenta) and counterstained with DAPI (grey). c , Quantification of the density of cleaved caspase-3 cells in P8 mice injected with hM4Di-mCherry and treated (magenta bar) or not treated (grey bar) with CNO between P5-P8 (2-tailed Student’s unpaired t -test, *** p =0.009, n = 8 animals for -CNO, and n = 7 animals for +CNO). d , Schematic of experimental design for CNO control experiments. e , Quantification of the density of PV (left) and SST (right) cells in P21 mice injected with hM3Dq-mCherry or hM4Di-mCherry and not treated with CNO (grey bars), or not injected with viruses and treated with CNO (magenta bars) between P5-P8 (ANOVA, p = 0.24 [PV] and p = 0.65 [SST] for PV, n = 7 animals for hM3Dq and hM4DI -CNO, 4 animals for non-injected +CNO; for SST, n = 9 animals for hM3Dq -CNO, 7 animals for hM4Di -CNO, and 4 animals for non-injected +CNO). Data is shown as mean ± SEM. Scale bar, 100 µm.

    Article Snippet: The following antibodies were used: goat anti-CTGF (1:200, Santa Cruz), rabbit anti cleaved-caspase-3 (1:200, Cell Signalling), rabbit anti-dsRed (1:500, Clontech), goat anti-mCherry (1:500, Antibodies-online), rabbit anti-GABA (1:2000, Millipore), mouse anti-GABA (1:500, Sigma), mouse anti-NeuN (1:500, Millipore) mouse anti-parvalbumin (1:1000, Swant), rabbit anti-parvalbumin (1:5000, Swant), rat anti-somatostatin (1:300, Millipore) and rabbit anti-PTEN (1:500, Abcam).

    Techniques: Mouse Assay, Injection

    Cleavage of K v 7.1 in physiology and pathophysiology.  a  Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1.  b  Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n  = 39 for vehicle,  n  = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n  = 27 for vehicle,  n  = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests.  c  Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls.  d  Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate.  e  Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test.  f  Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control.  c  Anti-KCNE1 antibody, anti-caspase 3 antibody.  c ,  d  Anti-K v 7.1 antibody, anti-GAPDH antibody.  e  Anti-β-actin antibody.  e ,  f  Anti-MYC antibody.  f  Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM

    Journal: Communications Biology

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    doi: 10.1038/s42003-018-0162-z

    Figure Lengend Snippet: Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n  = 39 for vehicle, n  = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n  = 27 for vehicle, n  = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM

    Article Snippet: The following antibodies were used: rabbit anti-β-actin (A2066, Sigma-Aldrich, St. Louis, USA), rabbit anti-caspase 3 (8G10, Cell Signaling, Cambridge, UK), rabbit anti-calmodulin (ab45689, Abcam, Cambridge, UK), rabbit anti-Eef2 (eukaryotic translation elongation factor 2, ab33523, Abcam, Cambridge, UK), mouse anti-GAPDH (MAB374, Millipore, Billerica, USA), rabbit anti-KCNE1 (APC-163, Alomone Labs, Jerusalem, Israel), rabbit anti-Kv 7.1 (ab77701, Abcam, Cambridge, UK), mouse anti-MYC (9B11, Cell Signaling, Cambridge, UK), goat anti-MYC (GTX29106, GeneTex Inc., Irvine, USA).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Binding Assay, Construct, Transfection, Mutagenesis, Negative Control

    Caspases are responsible for the generation of CTF2. a Western blot analysis of lysates derived from HEK 293T cells stably expressing K v 7.1-MYC treated with either 1 µmol per L staurosporine for 6 h or treated with 1 µmol per L staurosporine for 6 h and pretreated for 2 h with 20 or 50 µmol per L of a caspase-8 inhibitor II. Untransfected (Ø) and vehicle-treated cells served as negative controls. Densitometric analysis of 4–6 independent experiments of the CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. # indicates nonspecific binding of the antibody. b MCF-7 lysates expressing K v 7.1-MYC and caspase-3 or caspase-3-D28A-D175A analyzed by immunoblot. Untransfected (Ø) and eGFP-transfected cells served as negative controls. # indicates nonspecific binding of the antibody. c Lysates of HEK 293T cells stably expressing K v 7.1 and coexpressing indicated caspases analyzed by immunoblot. Untransfected (Ø) and eGFP-transfected cells served as negative controls. d Lysates of HL-1 cells treated for 6.5 h with 0.5, 1, 1.5 and 2 µmol per L of staurosporine analyzed by immunoblot. Vehicle-treated cell lysates served as negative control. Densitometric analysis of CTF2 band intensity normalized to Kv7.1 full-length band intensity of five independent experiments. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. a − c Anti-MYC antibody. Anti-β-actin antibody. a , b , d Anti-caspase-3 antibody. d Anti-K v 7.1 antibody. All dot blots are shown as mean and error bars as SEM

    Journal: Communications Biology

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    doi: 10.1038/s42003-018-0162-z

    Figure Lengend Snippet: Caspases are responsible for the generation of CTF2. a Western blot analysis of lysates derived from HEK 293T cells stably expressing K v 7.1-MYC treated with either 1 µmol per L staurosporine for 6 h or treated with 1 µmol per L staurosporine for 6 h and pretreated for 2 h with 20 or 50 µmol per L of a caspase-8 inhibitor II. Untransfected (Ø) and vehicle-treated cells served as negative controls. Densitometric analysis of 4–6 independent experiments of the CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. # indicates nonspecific binding of the antibody. b MCF-7 lysates expressing K v 7.1-MYC and caspase-3 or caspase-3-D28A-D175A analyzed by immunoblot. Untransfected (Ø) and eGFP-transfected cells served as negative controls. # indicates nonspecific binding of the antibody. c Lysates of HEK 293T cells stably expressing K v 7.1 and coexpressing indicated caspases analyzed by immunoblot. Untransfected (Ø) and eGFP-transfected cells served as negative controls. d Lysates of HL-1 cells treated for 6.5 h with 0.5, 1, 1.5 and 2 µmol per L of staurosporine analyzed by immunoblot. Vehicle-treated cell lysates served as negative control. Densitometric analysis of CTF2 band intensity normalized to Kv7.1 full-length band intensity of five independent experiments. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. a − c Anti-MYC antibody. Anti-β-actin antibody. a , b , d Anti-caspase-3 antibody. d Anti-K v 7.1 antibody. All dot blots are shown as mean and error bars as SEM

    Article Snippet: The following antibodies were used: rabbit anti-β-actin (A2066, Sigma-Aldrich, St. Louis, USA), rabbit anti-caspase 3 (8G10, Cell Signaling, Cambridge, UK), rabbit anti-calmodulin (ab45689, Abcam, Cambridge, UK), rabbit anti-Eef2 (eukaryotic translation elongation factor 2, ab33523, Abcam, Cambridge, UK), mouse anti-GAPDH (MAB374, Millipore, Billerica, USA), rabbit anti-KCNE1 (APC-163, Alomone Labs, Jerusalem, Israel), rabbit anti-Kv 7.1 (ab77701, Abcam, Cambridge, UK), mouse anti-MYC (9B11, Cell Signaling, Cambridge, UK), goat anti-MYC (GTX29106, GeneTex Inc., Irvine, USA).

    Techniques: Western Blot, Derivative Assay, Stable Transfection, Expressing, Binding Assay, Transfection, Negative Control

    Doxorubicin induces cleavage of Kv7.1 to CTF2. a Immunoblot analysis of human-induced pluripotent stem cell-derived cardiomyocytes treated either with vehicle, staurosporine (2 µM) for 4 h or doxorubicin (10 µM) overnight. # indicates nonspecific binding of the K v 7.1 antibody. b Densitometric analysis of 3–6 independent experiments of band intensities of CTF2 normalized to GAPDH band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. a Anti-K v 7.1 antibody, anti-caspase-3 antibody, anti-GAPDH antibody. All dot blots are shown as mean and error bars as SEM

    Journal: Communications Biology

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    doi: 10.1038/s42003-018-0162-z

    Figure Lengend Snippet: Doxorubicin induces cleavage of Kv7.1 to CTF2. a Immunoblot analysis of human-induced pluripotent stem cell-derived cardiomyocytes treated either with vehicle, staurosporine (2 µM) for 4 h or doxorubicin (10 µM) overnight. # indicates nonspecific binding of the K v 7.1 antibody. b Densitometric analysis of 3–6 independent experiments of band intensities of CTF2 normalized to GAPDH band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. a Anti-K v 7.1 antibody, anti-caspase-3 antibody, anti-GAPDH antibody. All dot blots are shown as mean and error bars as SEM

    Article Snippet: The following antibodies were used: rabbit anti-β-actin (A2066, Sigma-Aldrich, St. Louis, USA), rabbit anti-caspase 3 (8G10, Cell Signaling, Cambridge, UK), rabbit anti-calmodulin (ab45689, Abcam, Cambridge, UK), rabbit anti-Eef2 (eukaryotic translation elongation factor 2, ab33523, Abcam, Cambridge, UK), mouse anti-GAPDH (MAB374, Millipore, Billerica, USA), rabbit anti-KCNE1 (APC-163, Alomone Labs, Jerusalem, Israel), rabbit anti-Kv 7.1 (ab77701, Abcam, Cambridge, UK), mouse anti-MYC (9B11, Cell Signaling, Cambridge, UK), goat anti-MYC (GTX29106, GeneTex Inc., Irvine, USA).

    Techniques: Derivative Assay, Binding Assay

    Glucose deprivation enhances the apoptosis of HeLa cell induced by S1. Notes: HeLa cells were treated with 10 µM S1, EBSS, and 10 µM S1 and EBSS for 8 hours. ( A ) The expression level of Cyto C, caspase-3, and PARP-1 was determined by Western blot. ( B ) Quantification of Cyto C, caspase-3, and PARP-1 levels was shown as mean ± SD (n=6). * P

    Journal: Cancer Management and Research

    Article Title: Glucose deprivation promotes apoptotic response to S1 by enhancing autophagy in human cervical cancer cells

    doi: 10.2147/CMAR.S184180

    Figure Lengend Snippet: Glucose deprivation enhances the apoptosis of HeLa cell induced by S1. Notes: HeLa cells were treated with 10 µM S1, EBSS, and 10 µM S1 and EBSS for 8 hours. ( A ) The expression level of Cyto C, caspase-3, and PARP-1 was determined by Western blot. ( B ) Quantification of Cyto C, caspase-3, and PARP-1 levels was shown as mean ± SD (n=6). * P

    Article Snippet: The antibody against caspase-3 (1:500, rabbit) was purchased from Cell Signaling (Beverly, MA, USA).

    Techniques: Expressing, Western Blot

    WT1 immunostaining in fetal adrenal gland. WT1 immunoreactivity was located in the cytoplasm of cells of the fetal zone and in the adrenal capsule, with some strongly reactive capsular cells intermingled with non-reactive cells (F, fetal zone; D, definitive zone; C, adrenal capsule).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal adrenal gland. WT1 immunoreactivity was located in the cytoplasm of cells of the fetal zone and in the adrenal capsule, with some strongly reactive capsular cells intermingled with non-reactive cells (F, fetal zone; D, definitive zone; C, adrenal capsule).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in placenta. WT1 immunoreactivity was restricted to cytoplasm of muscular cells in the arterial wall (black arrow) and star-shaped cells in close relationship with the cytotrophoblast (red arrows).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in placenta. WT1 immunoreactivity was restricted to cytoplasm of muscular cells in the arterial wall (black arrow) and star-shaped cells in close relationship with the cytotrophoblast (red arrows).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in fetal liver. WT1 cytoplasmic immunoreactivity was observed in the of mesenchymal cells of the developing portal tracts (black arrows) and developing biliary structures (red arrows), including remnants of the ductal plate (green arrow).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal liver. WT1 cytoplasmic immunoreactivity was observed in the of mesenchymal cells of the developing portal tracts (black arrows) and developing biliary structures (red arrows), including remnants of the ductal plate (green arrow).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in fetal heart. Atrial cardiomyocytes (black arrow) showed higher cytoplasmic levels of WT1 immunostaining as compared to those observed in both ventricles (red arrow).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal heart. Atrial cardiomyocytes (black arrow) showed higher cytoplasmic levels of WT1 immunostaining as compared to those observed in both ventricles (red arrow).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in fetal central nervous system. WT1 was detected A) in the cytoplasm of Radial glia and in the axons localized in the cerebral cortex (VZ, ventricular zone; MZ, marginal zone) and B) in the cytoplasm of Radial Glia of the spinal cord.

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal central nervous system. WT1 was detected A) in the cytoplasm of Radial glia and in the axons localized in the cerebral cortex (VZ, ventricular zone; MZ, marginal zone) and B) in the cytoplasm of Radial Glia of the spinal cord.

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in fetal gut. WT1 was detected A) in the cytoplasm of lamina propria of the mucosa and in the fibrovascular axis of villi (black arrows); B) in the submucosa (red arrow).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal gut. WT1 was detected A) in the cytoplasm of lamina propria of the mucosa and in the fibrovascular axis of villi (black arrows); B) in the submucosa (red arrow).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in fetal arts. WT1 cytoplasmic immunoreactivity were observed A) in developing skeletal muscles and B) in the progenitor cells of the developing derma (black arrow).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal arts. WT1 cytoplasmic immunoreactivity were observed A) in developing skeletal muscles and B) in the progenitor cells of the developing derma (black arrow).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in fetal lung. WT1 was expressed in cytoplasm of mesenchymal progenitors surrounding branching epithelial structures (black arrows) and in small vessels inside the pulmonary scarcely differentiated mesenchyme (red arrow).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal lung. WT1 was expressed in cytoplasm of mesenchymal progenitors surrounding branching epithelial structures (black arrows) and in small vessels inside the pulmonary scarcely differentiated mesenchyme (red arrow).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    WT1 immunostaining in fetal kidney. WT1 immunoreactivity was detected A) in the developing glomeruli (black arrows) and in the nephrogenic zone located under the renal capsule (red arrow); B) in the cytoplasm of interstitial cells surrounding tubular structures (black arrow); C) in the cytoplasm and nuclei of podocyte precursors (black arrows) and along the developing basal membranes inside glomeruli (red arrow); and D) in the cytoplasm of Cap mesenchimal cells (black arrows) and in spindle cells located in the renal interstitium (red arrow).

    Journal: European Journal of Histochemistry : EJH

    Article Title: WT1 Expression in the Human Fetus During Development

    doi: 10.4081/ejh.2015.2499

    Figure Lengend Snippet: WT1 immunostaining in fetal kidney. WT1 immunoreactivity was detected A) in the developing glomeruli (black arrows) and in the nephrogenic zone located under the renal capsule (red arrow); B) in the cytoplasm of interstitial cells surrounding tubular structures (black arrow); C) in the cytoplasm and nuclei of podocyte precursors (black arrows) and along the developing basal membranes inside glomeruli (red arrow); and D) in the cytoplasm of Cap mesenchimal cells (black arrows) and in spindle cells located in the renal interstitium (red arrow).

    Article Snippet: Two serial 3 µm-thick sections were obtained from each paraffin block; after dewaxing and rehydrating, one of these was stained with hematoxylin-eosin, the other pre-treated for immunohistochemical analysis, with 10 min heat-induced epitope retrieval in buffer pH 9.00 (EnVisionTM FLEX Target Retrieval Solution High pH, Code: K8004; Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Immunostaining

    Antrocin inhibits the PI3K/AKT and MAPK signaling pathways and phosphorylation of IGF-1R. PC3-KD cells were treated with IR (2 Gy), antrocin (100 μM), or IR plus antrocin, followed by incubation for 48 h. ( A ) The represented gene network was identified by ingenuity pathway analysis. The color of each line indicates the regulation of gene expression. ( B ) Cell lysates were harvested and subjected to Western blot analysis using antibodies against p85, p110, p-AKT, AKT, GSK3-β, p-GSK3-β, p-β-catenin, and β-catenin, and ( C ) antibodies against IGF-1R, p38, JNK, Erk1/2, and their respective phosphorylated forms. The data represent one of three independent experiments. β-actin was used as a loading control. The expression level of each protein was quantified by the signal intensity and normalized with each vehicle untreated group. The relative level of each protein expression was indicated at the bottom of each lane.

    Journal: Cancers

    Article Title: Antrocin Sensitizes Prostate Cancer Cells to Radiotherapy through Inhibiting PI3K/AKT and MAPK Signaling Pathways

    doi: 10.3390/cancers11010034

    Figure Lengend Snippet: Antrocin inhibits the PI3K/AKT and MAPK signaling pathways and phosphorylation of IGF-1R. PC3-KD cells were treated with IR (2 Gy), antrocin (100 μM), or IR plus antrocin, followed by incubation for 48 h. ( A ) The represented gene network was identified by ingenuity pathway analysis. The color of each line indicates the regulation of gene expression. ( B ) Cell lysates were harvested and subjected to Western blot analysis using antibodies against p85, p110, p-AKT, AKT, GSK3-β, p-GSK3-β, p-β-catenin, and β-catenin, and ( C ) antibodies against IGF-1R, p38, JNK, Erk1/2, and their respective phosphorylated forms. The data represent one of three independent experiments. β-actin was used as a loading control. The expression level of each protein was quantified by the signal intensity and normalized with each vehicle untreated group. The relative level of each protein expression was indicated at the bottom of each lane.

    Article Snippet: The sections were then stained with monoclonal antibodies against cyclin-D1, cleaved PARP, cleaved-caspase 9, phospho-AKT, and phospho-β-catenin (Cell Signaling) for 24 h at 4 °C.

    Techniques: Incubation, Expressing, Western Blot

    Representative immunohistochemical (IHC) staining patterns in xenograft PCa tissue. IHC analysis of paraffin sections show staining with specific antibodies against PARP, cleaved-caspase 9, phospho-AKT, phospho-β-catenin, and cyclin D1, respectively. Images were photographed at 200× magnification.

    Journal: Cancers

    Article Title: Antrocin Sensitizes Prostate Cancer Cells to Radiotherapy through Inhibiting PI3K/AKT and MAPK Signaling Pathways

    doi: 10.3390/cancers11010034

    Figure Lengend Snippet: Representative immunohistochemical (IHC) staining patterns in xenograft PCa tissue. IHC analysis of paraffin sections show staining with specific antibodies against PARP, cleaved-caspase 9, phospho-AKT, phospho-β-catenin, and cyclin D1, respectively. Images were photographed at 200× magnification.

    Article Snippet: The sections were then stained with monoclonal antibodies against cyclin-D1, cleaved PARP, cleaved-caspase 9, phospho-AKT, and phospho-β-catenin (Cell Signaling) for 24 h at 4 °C.

    Techniques: Immunohistochemistry, Staining

    Decrease in endogenous PAK1, Akt, and BAD phosphorylation in MCF10A cells grown in suspension. MCF10A cells were grown either attached (t = 0) or suspended for 2, 4, or 8 hours. (A) Western blots of whole cell lysates were probed with anti-phospho Akt

    Journal:

    Article Title: Active p21-Activated Kinase 1 Rescues MCF10A Breast Epithelial Cells from Undergoing Anoikis 1

    doi:

    Figure Lengend Snippet: Decrease in endogenous PAK1, Akt, and BAD phosphorylation in MCF10A cells grown in suspension. MCF10A cells were grown either attached (t = 0) or suspended for 2, 4, or 8 hours. (A) Western blots of whole cell lysates were probed with anti-phospho Akt

    Article Snippet: Antibodies to phosphoPAK1 (Thr-423)/PAK2 (Thr-402), phosphoAkt (Ser-423), total Akt, total BAD, phosphoBAD (Ser-112 and Ser-136), and phospho-p70 S6 kinase (Thr-421 and Ser-424) were purchased from Cell Signalling Technology (Beverly, MA).

    Techniques: Western Blot

    Effects of melatonin on the activation of the PI3K/AKT and MAPK- ERK1/2 pathways(A). Effect of inhibition of LY294002 and wortmannin on the activation of the PI3K/AKT pathway (B, C). Effect of MTR inhibitors on the activation of the PI3K/AKT pathway (D) in the TI in small follicles. Small follicles were treated with 10 ng/mL melatonin for 0 min, 30 min, 1 h, or 4 h with or without pretreatment with a combination of inhibitors, 25 μM LY294002, 0.1 μM wortmannin, 10 μM luzindole, or 10 μM 4P-PDOT for 30 min. TI lysates were subjected to SDS-PAGE/immunoblotting for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 analysis. The results are the mean ± SEM of three independent experiments. The relative density ratio was calculated on the basis of a control group value of one. * P

    Journal: International Journal of Biological Sciences

    Article Title: Melatonin Stimulates STAR Expression and Progesterone Production via Activation of the PI3K/AKT Pathway in Bovine Theca Cells

    doi: 10.7150/ijbs.27912

    Figure Lengend Snippet: Effects of melatonin on the activation of the PI3K/AKT and MAPK- ERK1/2 pathways(A). Effect of inhibition of LY294002 and wortmannin on the activation of the PI3K/AKT pathway (B, C). Effect of MTR inhibitors on the activation of the PI3K/AKT pathway (D) in the TI in small follicles. Small follicles were treated with 10 ng/mL melatonin for 0 min, 30 min, 1 h, or 4 h with or without pretreatment with a combination of inhibitors, 25 μM LY294002, 0.1 μM wortmannin, 10 μM luzindole, or 10 μM 4P-PDOT for 30 min. TI lysates were subjected to SDS-PAGE/immunoblotting for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 analysis. The results are the mean ± SEM of three independent experiments. The relative density ratio was calculated on the basis of a control group value of one. * P

    Article Snippet: Anti-total-AKT, anti-phospho-AKT, anti-total-ERK1/2 and anti-phospho-ERK1/2 antibodies were from Cell Signaling Technology, Inc (Beverly, MA).

    Techniques: Activation Assay, Inhibition, SDS Page

    Ghrelin expression in node negative breast cancer tissue was analyzed by immunohistochemistry. Representative images of ghrelin with 0 (non-immunoreactive), 1 (weak), 2 (moderate) and 3 (strong) immunostaining. Scale bar = 100 μm.

    Journal: PLoS ONE

    Article Title: Ghrelin is a prognostic marker and a potential therapeutic target in breast cancer

    doi: 10.1371/journal.pone.0176059

    Figure Lengend Snippet: Ghrelin expression in node negative breast cancer tissue was analyzed by immunohistochemistry. Representative images of ghrelin with 0 (non-immunoreactive), 1 (weak), 2 (moderate) and 3 (strong) immunostaining. Scale bar = 100 μm.

    Article Snippet: Immunohistochemistry Immunohistochemical staining was performed using the Dako EnVision Plus-HRP Detection Kit (K401111-2, Dako, Glostrup, Denmark) according to the manufacturer’s instructions.

    Techniques: Expressing, Immunohistochemistry, Immunostaining

    Etoposide inhibits the EMT/β-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear β-catenin downregulation. a Effect of TOP2 isoforms knockdown on the expression of EMT markers, STT3 and PD-L1 in 4T1 cell. b Representative phase-contrast microscopy images of 4T1 cell treated with indicated sg-RNAs. Scale bar, 50 μm. c Influence of TOP2 isoforms knockdown on etoposide-induced MET and downregulation of STT3 and PD-L1. d Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated cancer cell killing assay (bottom right) of tumorspheres cultured from various sgRNA-treated 4T1 cells. e Effect of proteasome inhibitor (MG132) and caspase inhibitor (Z-VAD-FMK) on etoposide-induced TOP2B degradation, MET, and downregulation of STT3 and PD-L1. f Western blotting of whole-cell extracts analyzing the effect of etoposide and TOP2B knockdown on total and active (non-phospho) β-catenin (β-Cat). g Western blotting of whole-cell extracts (WCE), cytosolic (Cyto), membrane (Mem), or nuclear (Nuc) fractions from sgRNA-treated 4T1 cells analyzing the effect of TOP2B knockdown on β-catenin subcellular localizations. Histone H3, E-cadherin (E-Cad), and tubulin were used as makers of nuclear, membrane, and cytosolic fractions, respectively. h Efficacy of β-catenin knockdown (si-β-Cat) in desensitizing cells to etoposide- and sg-TOP2B- induced MET and downregulation of STT3 and PD-L1. Nuc nuclear fraction, WCE whole-cell extracts. i Co-immunoprecipitation assay showing interaction between TOP2B and β-catenin. j Duolink assay analyzing the interaction of TOP2B and active (non-phospho) β-catenin in the nucleus. Red dots (indicated by arrows) represented TOP2B-β-catenin interaction signals and nuclei were counterstained with DAPI (blue). The right column shows higher-magnification image of the area outlined in the left column. Scale bar, 5 μm. Error bars represent s.d. ( n = 3). * P

    Journal: Nature Communications

    Article Title: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion

    doi: 10.1038/s41467-018-04313-6

    Figure Lengend Snippet: Etoposide inhibits the EMT/β-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear β-catenin downregulation. a Effect of TOP2 isoforms knockdown on the expression of EMT markers, STT3 and PD-L1 in 4T1 cell. b Representative phase-contrast microscopy images of 4T1 cell treated with indicated sg-RNAs. Scale bar, 50 μm. c Influence of TOP2 isoforms knockdown on etoposide-induced MET and downregulation of STT3 and PD-L1. d Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated cancer cell killing assay (bottom right) of tumorspheres cultured from various sgRNA-treated 4T1 cells. e Effect of proteasome inhibitor (MG132) and caspase inhibitor (Z-VAD-FMK) on etoposide-induced TOP2B degradation, MET, and downregulation of STT3 and PD-L1. f Western blotting of whole-cell extracts analyzing the effect of etoposide and TOP2B knockdown on total and active (non-phospho) β-catenin (β-Cat). g Western blotting of whole-cell extracts (WCE), cytosolic (Cyto), membrane (Mem), or nuclear (Nuc) fractions from sgRNA-treated 4T1 cells analyzing the effect of TOP2B knockdown on β-catenin subcellular localizations. Histone H3, E-cadherin (E-Cad), and tubulin were used as makers of nuclear, membrane, and cytosolic fractions, respectively. h Efficacy of β-catenin knockdown (si-β-Cat) in desensitizing cells to etoposide- and sg-TOP2B- induced MET and downregulation of STT3 and PD-L1. Nuc nuclear fraction, WCE whole-cell extracts. i Co-immunoprecipitation assay showing interaction between TOP2B and β-catenin. j Duolink assay analyzing the interaction of TOP2B and active (non-phospho) β-catenin in the nucleus. Red dots (indicated by arrows) represented TOP2B-β-catenin interaction signals and nuclei were counterstained with DAPI (blue). The right column shows higher-magnification image of the area outlined in the left column. Scale bar, 5 μm. Error bars represent s.d. ( n = 3). * P

    Article Snippet: Antibodies used for tissue staining were as follows: PD-L1 (Cell Signaling, #64988, 1:200), CD8 (marker for cytotoxic T lymphocyte; abcam, #ab22378, 1:200), and granzyme b (marker for activated cytotoxic T lymphocyte; R & D Systems, #AF1865, 1:100).

    Techniques: Expressing, Microscopy, Western Blot, Binding Assay, In Vitro, Cell Culture, Co-Immunoprecipitation Assay

    Etoposide inhibits the EMT/β-catenin/STT3/PD-L1 axis and downregulates PD-L1 of CSC and non-CSC populations. a Effect of etoposide (ETO) on the protein and mRNA expression of EMT markers, STT3, and PD-L1 in the general cell population of 4T1 cells. E-Cad E-cadherin, N-Cad N-cadherin, Vim vimentin. b Effect of etoposide and exogenous STT3A/B on PD-L1 expression in the general cell population of 4T1 cells. c Flow cytometric analysis of the influence of etoposide and exogenous STT3A/B on the frequency (left) and PD-L1 expression (right) of 4T1 CSC (CD44 + CD24 + ALDH1 + ) populations. Open histograms represent isotype IgG negative control. d Experimental workflow of preparing spheres derived from etoposide-treated 4T1 cells. e Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated tumor cell killing assay (bottom right) of tumorspheres derived from 4T1 cells treated with etoposide and exogenous STT3A/B. f – h Tumor-seeding ability ( f ), tumor onset curve ( g ), and tumor growth curve ( h ) of tumorspheres cultured from etoposide-treated 4T1 cells. i Representative images of tumors staining with PD-L1, CD8, granzyme b (GZMB), and Hoechst. Scale bar, 200 μm. The intensity of immunofluorescence signal was quantified from nine core biopsy sections, normalized relative to the control group, and shown as means ± s.d. Other error bars represent s.d. ( n = 3). * P

    Journal: Nature Communications

    Article Title: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion

    doi: 10.1038/s41467-018-04313-6

    Figure Lengend Snippet: Etoposide inhibits the EMT/β-catenin/STT3/PD-L1 axis and downregulates PD-L1 of CSC and non-CSC populations. a Effect of etoposide (ETO) on the protein and mRNA expression of EMT markers, STT3, and PD-L1 in the general cell population of 4T1 cells. E-Cad E-cadherin, N-Cad N-cadherin, Vim vimentin. b Effect of etoposide and exogenous STT3A/B on PD-L1 expression in the general cell population of 4T1 cells. c Flow cytometric analysis of the influence of etoposide and exogenous STT3A/B on the frequency (left) and PD-L1 expression (right) of 4T1 CSC (CD44 + CD24 + ALDH1 + ) populations. Open histograms represent isotype IgG negative control. d Experimental workflow of preparing spheres derived from etoposide-treated 4T1 cells. e Western blot analysis (left), PD-1 binding assay (upper right), and in vitro PBMC-mediated tumor cell killing assay (bottom right) of tumorspheres derived from 4T1 cells treated with etoposide and exogenous STT3A/B. f – h Tumor-seeding ability ( f ), tumor onset curve ( g ), and tumor growth curve ( h ) of tumorspheres cultured from etoposide-treated 4T1 cells. i Representative images of tumors staining with PD-L1, CD8, granzyme b (GZMB), and Hoechst. Scale bar, 200 μm. The intensity of immunofluorescence signal was quantified from nine core biopsy sections, normalized relative to the control group, and shown as means ± s.d. Other error bars represent s.d. ( n = 3). * P

    Article Snippet: Antibodies used for tissue staining were as follows: PD-L1 (Cell Signaling, #64988, 1:200), CD8 (marker for cytotoxic T lymphocyte; abcam, #ab22378, 1:200), and granzyme b (marker for activated cytotoxic T lymphocyte; R & D Systems, #AF1865, 1:100).

    Techniques: Expressing, Flow Cytometry, Negative Control, Derivative Assay, Western Blot, Binding Assay, In Vitro, Cell Culture, Staining, Immunofluorescence

    Etoposide enhances the therapeutic efficacy of Tim-3 blockade therapy. a Schematic diagram illustrating the treatment protocol of Tim-3 antibody (Tim-3 Ab) and/or etoposide (ETO) in mice. At the endpoint, tumor cells and tumor-infiltrating lymphocytes (TIL) were isolated for analysis. b Tumor growth of 4T1 cells in BALB/c mice treated with Tim-3 antibody and/or etoposide. Tumor size was measured at the indicated time points and tumor weight was measured at the endpoint ( n = 6 mice per group). c Efficacy of indicated treatments on the CSC (CD44 + CD24 + ALDH1 + populations) frequency of 4T1 tumors. d Flow cytometric analysis analyzing the effect of indicated treatments on the expression of E-cadherin (E-Cad, epithelial marker) and N-cadherin (N-Cad, mesenchymal marker) in the entire cancer cell population of 4T1 tumors. e qRT-PCR analysis of the influence of indicated treatments on STT3 isoforms expression in the entire cancer cell population of 4T1 tumors. f Effect of indicated treatments on PD-L1 expression in the CSC and non-CSC populations of 4T1 tumors. g Intracellular cytokine staining of CD8 + IFNγ + cells in the CD3 + T cell populations from isolated tumor-infiltrating lymphocytes to analyze the impacts of indicated treatments on tumor-infiltrating cytotoxic T cell activity. Error bars represent s.d. ( n = 6). * P

    Journal: Nature Communications

    Article Title: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion

    doi: 10.1038/s41467-018-04313-6

    Figure Lengend Snippet: Etoposide enhances the therapeutic efficacy of Tim-3 blockade therapy. a Schematic diagram illustrating the treatment protocol of Tim-3 antibody (Tim-3 Ab) and/or etoposide (ETO) in mice. At the endpoint, tumor cells and tumor-infiltrating lymphocytes (TIL) were isolated for analysis. b Tumor growth of 4T1 cells in BALB/c mice treated with Tim-3 antibody and/or etoposide. Tumor size was measured at the indicated time points and tumor weight was measured at the endpoint ( n = 6 mice per group). c Efficacy of indicated treatments on the CSC (CD44 + CD24 + ALDH1 + populations) frequency of 4T1 tumors. d Flow cytometric analysis analyzing the effect of indicated treatments on the expression of E-cadherin (E-Cad, epithelial marker) and N-cadherin (N-Cad, mesenchymal marker) in the entire cancer cell population of 4T1 tumors. e qRT-PCR analysis of the influence of indicated treatments on STT3 isoforms expression in the entire cancer cell population of 4T1 tumors. f Effect of indicated treatments on PD-L1 expression in the CSC and non-CSC populations of 4T1 tumors. g Intracellular cytokine staining of CD8 + IFNγ + cells in the CD3 + T cell populations from isolated tumor-infiltrating lymphocytes to analyze the impacts of indicated treatments on tumor-infiltrating cytotoxic T cell activity. Error bars represent s.d. ( n = 6). * P

    Article Snippet: Antibodies used for tissue staining were as follows: PD-L1 (Cell Signaling, #64988, 1:200), CD8 (marker for cytotoxic T lymphocyte; abcam, #ab22378, 1:200), and granzyme b (marker for activated cytotoxic T lymphocyte; R & D Systems, #AF1865, 1:100).

    Techniques: Mouse Assay, Isolation, Flow Cytometry, Expressing, Marker, Quantitative RT-PCR, Staining, Activity Assay

    EMT induces a more robust upregulation of PD-L1 protein in CSCs than in non-CSCs without significant induction of PD-L1 mRNA levels. a , c Western blot analysis of PD-L1 and EMT markers in the general cell population of MCF-10A cells undergoing EMT driven by TGF-β ( a ) or RasV12 ( c ). E-Cad E-cadherin, N-Cad N-cadherin, Vim vimentin. b , d Flow cytometric analysis of PD-L1 protein expression in breast stem-like cell (SC) (CD44 + CD24 −/low , red histograms) and non-SC (non-CD44 + CD24 −/low , blue histograms) populations from control cells and EMT cells driven by TGF-β ( b ) or RasV12 ( d ). Open histograms represent isotype IgG negative control. The mean fluorescence intensity (MFI) of each cell population was quantified by FlowJo and compared. e , f Representative phase-contrast microscopy images and PD-L1 expression of spheres growing from control or EMT cells driven by TGF-β ( e ) or RasV12 ( f ). Scale bar, 200 μm. g , h qRT-PCR analysis comparing PD-L1 (CD274) mRNA induction levels between SC (CD44 + CD24 −/low ) and non-SC (non-CD44 + CD24 −/low ) populations upon TGF-β ( g ) or RasV12 ( h ) treatment. Error bars represent s.d. ( n = 3). * P

    Journal: Nature Communications

    Article Title: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion

    doi: 10.1038/s41467-018-04313-6

    Figure Lengend Snippet: EMT induces a more robust upregulation of PD-L1 protein in CSCs than in non-CSCs without significant induction of PD-L1 mRNA levels. a , c Western blot analysis of PD-L1 and EMT markers in the general cell population of MCF-10A cells undergoing EMT driven by TGF-β ( a ) or RasV12 ( c ). E-Cad E-cadherin, N-Cad N-cadherin, Vim vimentin. b , d Flow cytometric analysis of PD-L1 protein expression in breast stem-like cell (SC) (CD44 + CD24 −/low , red histograms) and non-SC (non-CD44 + CD24 −/low , blue histograms) populations from control cells and EMT cells driven by TGF-β ( b ) or RasV12 ( d ). Open histograms represent isotype IgG negative control. The mean fluorescence intensity (MFI) of each cell population was quantified by FlowJo and compared. e , f Representative phase-contrast microscopy images and PD-L1 expression of spheres growing from control or EMT cells driven by TGF-β ( e ) or RasV12 ( f ). Scale bar, 200 μm. g , h qRT-PCR analysis comparing PD-L1 (CD274) mRNA induction levels between SC (CD44 + CD24 −/low ) and non-SC (non-CD44 + CD24 −/low ) populations upon TGF-β ( g ) or RasV12 ( h ) treatment. Error bars represent s.d. ( n = 3). * P

    Article Snippet: Antibodies used for tissue staining were as follows: PD-L1 (Cell Signaling, #64988, 1:200), CD8 (marker for cytotoxic T lymphocyte; abcam, #ab22378, 1:200), and granzyme b (marker for activated cytotoxic T lymphocyte; R & D Systems, #AF1865, 1:100).

    Techniques: Western Blot, Flow Cytometry, Expressing, Negative Control, Fluorescence, Microscopy, Quantitative RT-PCR

    STT3-dependent PD-L1 glycosylation is sufficient and required for effective EMT-mediated PD-L1 induction in the general cell population. a , b Protein and mRNA induction of STT3A, STT3B, and PD-L1 (CD274) in the general cell population of MCF-10A cells undergoing EMT driven by TGF-β ( a ) or RasV12 ( b ). c Effect of ectopic expression of STT3 isoforms on endogenous PD-L1 protein and mRNA induction in the general cell population of MCF-10A cells without TGF-β treatment. d Influence of endogenous STT3 isoforms knockdown on TGF-β-mediated PD-L1 protein induction and PD-L1 molecular weight; s.e. short exposure, l.e. long exposure. e , f Flow cytometric analysis comparing cell surface PD-L1 expression ( e ) and PD-1 binding abilities ( f ) between various sgRNA-transfected MCF-10A cells after TGF-β treatment. g qRT-PCR analysis of PD-L1 mRNA levels in STT3 knockdown MCF-10A cells upon TGF-β treatment. h ConA lectin binding assay analyzing the glycosylation status of PD-L1 protein purified from STT3 knockdown cells. i Cycloheximide (CHX) chase assay of PD-L1 protein turnover rates in STT3 knockdown cells. j Top: Schematic illustrating co-expression constructs of PD-L1 (wt or 4NQ) and green fluorescent protein (GFP) used to assay the protein expression status of PD-L1. GFP was used as an internal control for transfection efficiency and gene expression. IRES internal ribosome entry site. Bottom: effect of ectopic STT3 isoforms on the protein expression amounts of wild-type (wt) PD-L1 and glycosylation-site mutant (4NQ). Error bars represent s.d. ( n = 3). * P

    Journal: Nature Communications

    Article Title: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion

    doi: 10.1038/s41467-018-04313-6

    Figure Lengend Snippet: STT3-dependent PD-L1 glycosylation is sufficient and required for effective EMT-mediated PD-L1 induction in the general cell population. a , b Protein and mRNA induction of STT3A, STT3B, and PD-L1 (CD274) in the general cell population of MCF-10A cells undergoing EMT driven by TGF-β ( a ) or RasV12 ( b ). c Effect of ectopic expression of STT3 isoforms on endogenous PD-L1 protein and mRNA induction in the general cell population of MCF-10A cells without TGF-β treatment. d Influence of endogenous STT3 isoforms knockdown on TGF-β-mediated PD-L1 protein induction and PD-L1 molecular weight; s.e. short exposure, l.e. long exposure. e , f Flow cytometric analysis comparing cell surface PD-L1 expression ( e ) and PD-1 binding abilities ( f ) between various sgRNA-transfected MCF-10A cells after TGF-β treatment. g qRT-PCR analysis of PD-L1 mRNA levels in STT3 knockdown MCF-10A cells upon TGF-β treatment. h ConA lectin binding assay analyzing the glycosylation status of PD-L1 protein purified from STT3 knockdown cells. i Cycloheximide (CHX) chase assay of PD-L1 protein turnover rates in STT3 knockdown cells. j Top: Schematic illustrating co-expression constructs of PD-L1 (wt or 4NQ) and green fluorescent protein (GFP) used to assay the protein expression status of PD-L1. GFP was used as an internal control for transfection efficiency and gene expression. IRES internal ribosome entry site. Bottom: effect of ectopic STT3 isoforms on the protein expression amounts of wild-type (wt) PD-L1 and glycosylation-site mutant (4NQ). Error bars represent s.d. ( n = 3). * P

    Article Snippet: Antibodies used for tissue staining were as follows: PD-L1 (Cell Signaling, #64988, 1:200), CD8 (marker for cytotoxic T lymphocyte; abcam, #ab22378, 1:200), and granzyme b (marker for activated cytotoxic T lymphocyte; R & D Systems, #AF1865, 1:100).

    Techniques: Expressing, Molecular Weight, Flow Cytometry, Binding Assay, Transfection, Quantitative RT-PCR, Purification, Construct, Mutagenesis

    EMT transcriptionally induces STT3 through β-catenin/TCF4. a ENCODE data display ChIP-seq signals for the occupancy of TCF4 (TCF7L2), histone H3K4Me3, and DNase Clusters around the transcription start sites of STT3A and STT3B. H3K4Me3 and DNase Clusters were used to define the transcriptional regulatory regions of STT3A and STT3B. These ENCODE data were generated by the ENCODE consortium and available on the Genome Browser at UCSC. b Pearson correlation analysis of β-catenin (CTNNB1) with STT3A and STT3B in TCGA breast cancer dataset ( n = 1100). c Top: schematic presentation of the wild-type (wt) and TCF4-binding-site-mutated (mt) STT3 promoter-luciferase reporter constructs of STT3A (−1042/+210, related to the transcription start site) and STT3B (−1381/−1, related to the transcription start site). The TCF4 binding sites on the promoter regions of STT3A ( −282 GCAAACCGACA −272 ) or STT3B (−889 TCACGTGGTGA −879 , −671 TCTTTCAACTG −661 ) are shown. Bottom: promoter luciferase activity in response to β-catenin (β-Cat) and TCF4 dominant-negative (TCF4-DN) mutant. d Effect of β-catenin (β-Cat) and TCF4-DN on the protein and mRNA expression of STT3 isoforms in MCF-10A cells. e Influence of STT3 knockdown on β-catenin (β-Cat)-mediated PD-L1 induction in MCF-10A cells; s.e. short exposure, l.e. long exposure. f Effect of β-catenin knockdown (si-β-Cat) and TCF4-DN on TGF-β-mediated PD-L1 induction in MCF-10A cells. g Effect of β-catenin knockdown (si-β-Cat) on the expression of STTA/B and PD-L1 in mesenchymal-like BT-549 cells. Error bars represent s.d. ( n = 3). * P

    Journal: Nature Communications

    Article Title: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion

    doi: 10.1038/s41467-018-04313-6

    Figure Lengend Snippet: EMT transcriptionally induces STT3 through β-catenin/TCF4. a ENCODE data display ChIP-seq signals for the occupancy of TCF4 (TCF7L2), histone H3K4Me3, and DNase Clusters around the transcription start sites of STT3A and STT3B. H3K4Me3 and DNase Clusters were used to define the transcriptional regulatory regions of STT3A and STT3B. These ENCODE data were generated by the ENCODE consortium and available on the Genome Browser at UCSC. b Pearson correlation analysis of β-catenin (CTNNB1) with STT3A and STT3B in TCGA breast cancer dataset ( n = 1100). c Top: schematic presentation of the wild-type (wt) and TCF4-binding-site-mutated (mt) STT3 promoter-luciferase reporter constructs of STT3A (−1042/+210, related to the transcription start site) and STT3B (−1381/−1, related to the transcription start site). The TCF4 binding sites on the promoter regions of STT3A ( −282 GCAAACCGACA −272 ) or STT3B (−889 TCACGTGGTGA −879 , −671 TCTTTCAACTG −661 ) are shown. Bottom: promoter luciferase activity in response to β-catenin (β-Cat) and TCF4 dominant-negative (TCF4-DN) mutant. d Effect of β-catenin (β-Cat) and TCF4-DN on the protein and mRNA expression of STT3 isoforms in MCF-10A cells. e Influence of STT3 knockdown on β-catenin (β-Cat)-mediated PD-L1 induction in MCF-10A cells; s.e. short exposure, l.e. long exposure. f Effect of β-catenin knockdown (si-β-Cat) and TCF4-DN on TGF-β-mediated PD-L1 induction in MCF-10A cells. g Effect of β-catenin knockdown (si-β-Cat) on the expression of STTA/B and PD-L1 in mesenchymal-like BT-549 cells. Error bars represent s.d. ( n = 3). * P

    Article Snippet: Antibodies used for tissue staining were as follows: PD-L1 (Cell Signaling, #64988, 1:200), CD8 (marker for cytotoxic T lymphocyte; abcam, #ab22378, 1:200), and granzyme b (marker for activated cytotoxic T lymphocyte; R & D Systems, #AF1865, 1:100).

    Techniques: Chromatin Immunoprecipitation, Generated, Binding Assay, Luciferase, Construct, Activity Assay, Dominant Negative Mutation, Mutagenesis, Expressing

    EMT induces higher levels of STT3 in CSCs than in non-CSCs, resulting in enriched PD-L1 in CSCs. a , b qRT-PCR and western blot analyses of STT3 induction in SCs (CD44 + CD24 −/low populations) and non-SCs (non-CD44 + CD24 −/low populations) of MCF-10A after TGF-β ( a ) or RasV12 ( b ) treatment. c qRT-PCR and western blot analyses of intrinsic STT3 expression in CSCs (CD44 + CD24 −/low populations) and non-CSCs (non-CD44 + CD24 −/low populations) of BT-549 cells. d Flow cytometric analysis of the effect of STT3A/3B knockdown on PD-L1 expression in CSCs and non-CSCs. e Efficacy of STT3A/3B knockdown in sensitizing CSCs to PBMC-mediated cancer cell killing. Error bars represent s.d. ( n = 3). * P

    Journal: Nature Communications

    Article Title: STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion

    doi: 10.1038/s41467-018-04313-6

    Figure Lengend Snippet: EMT induces higher levels of STT3 in CSCs than in non-CSCs, resulting in enriched PD-L1 in CSCs. a , b qRT-PCR and western blot analyses of STT3 induction in SCs (CD44 + CD24 −/low populations) and non-SCs (non-CD44 + CD24 −/low populations) of MCF-10A after TGF-β ( a ) or RasV12 ( b ) treatment. c qRT-PCR and western blot analyses of intrinsic STT3 expression in CSCs (CD44 + CD24 −/low populations) and non-CSCs (non-CD44 + CD24 −/low populations) of BT-549 cells. d Flow cytometric analysis of the effect of STT3A/3B knockdown on PD-L1 expression in CSCs and non-CSCs. e Efficacy of STT3A/3B knockdown in sensitizing CSCs to PBMC-mediated cancer cell killing. Error bars represent s.d. ( n = 3). * P

    Article Snippet: Antibodies used for tissue staining were as follows: PD-L1 (Cell Signaling, #64988, 1:200), CD8 (marker for cytotoxic T lymphocyte; abcam, #ab22378, 1:200), and granzyme b (marker for activated cytotoxic T lymphocyte; R & D Systems, #AF1865, 1:100).

    Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry