anti gapdh antibody Search Results


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  • 99
    Thermo Fisher anti gapdh
    Anti Gapdh, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1766 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gapdh
    Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7859 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology gapdh
    ( A and B ) Local injection of <t>emerin</t> ( A ) or lamin A ( B ) lentiviruses (lenti) significantly increased the expression of emerin or lamin A/C in common carotid arteries the hypertensive rats. ( C ) Immunofluorescence staining against BrdU revealed that locally injected lentiviruses of emerin or lamin A remarkably decreased VSMC proliferation in the media of common carotid arteries of hypertensive rats. For Western blot, <t>GAPDH</t> was used for normalization. Values are expressed as mean ± SD. ** P
    Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 33667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gapdh
    ( A and B ) Local injection of <t>emerin</t> ( A ) or lamin A ( B ) lentiviruses (lenti) significantly increased the expression of emerin or lamin A/C in common carotid arteries the hypertensive rats. ( C ) Immunofluorescence staining against BrdU revealed that locally injected lentiviruses of emerin or lamin A remarkably decreased VSMC proliferation in the media of common carotid arteries of hypertensive rats. For Western blot, <t>GAPDH</t> was used for normalization. Values are expressed as mean ± SD. ** P
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 30834 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gapdh  (Abcam)
    99
    Abcam gapdh
    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone <t>H3</t> served as the nuclear loading control, <t>GAPDH</t> served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P
    Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 18714 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti gapdh
    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone <t>H3</t> served as the nuclear loading control, <t>GAPDH</t> served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P
    Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 10431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh
    TCR signaling induces RORγt phosphorylation and subsequent <t>AhR-RORγt</t> interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and <t>GAPDH</t> proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.
    Anti Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8036 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gapdh
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16774 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti gapdh
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Mouse Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh antibody
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Anti Gapdh Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gapdh antibody mouse monoclonal
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Anti Gapdh Antibody Mouse Monoclonal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3458 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh antibody 6c5
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Anti Gapdh Antibody 6c5, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh antibody epr16891
    Western blot analysis of transforming growth factor beta <t>(TGF‐β)</t> and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase <t>(GAPDH)</t> antibodies. (b) Arbitrary values expressed as mean and SD. * P
    Anti Gapdh Antibody Epr16891, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 737 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse anti gapdh
    Kidney-replenishing herb promoted <t>SOCS-3</t> protein expression in human first-trimester trophoblasts. Primary human trophoblasts were incubated for 4 h with 10% to 20% Kidney-replenishing herb. As shown in (a), (b) there is also an increase of SOCS-3 protein expression (0.5, 1, 2, and 4 h). Control cells were treated with serum in the same way without herb and detected a trace SOCS-3 expression at 2 h (c). The levels of <t>GAPDH</t> were examined as an internal control (d). The experiments were performed at least three times and representative result was presented.
    Mouse Anti Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1920 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh antibody loading control
    Kidney-replenishing herb promoted <t>SOCS-3</t> protein expression in human first-trimester trophoblasts. Primary human trophoblasts were incubated for 4 h with 10% to 20% Kidney-replenishing herb. As shown in (a), (b) there is also an increase of SOCS-3 protein expression (0.5, 1, 2, and 4 h). Control cells were treated with serum in the same way without herb and detected a trace SOCS-3 expression at 2 h (c). The levels of <t>GAPDH</t> were examined as an internal control (d). The experiments were performed at least three times and representative result was presented.
    Anti Gapdh Antibody Loading Control, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gapdh antibody
    Kidney-replenishing herb promoted <t>SOCS-3</t> protein expression in human first-trimester trophoblasts. Primary human trophoblasts were incubated for 4 h with 10% to 20% Kidney-replenishing herb. As shown in (a), (b) there is also an increase of SOCS-3 protein expression (0.5, 1, 2, and 4 h). Control cells were treated with serum in the same way without herb and detected a trace SOCS-3 expression at 2 h (c). The levels of <t>GAPDH</t> were examined as an internal control (d). The experiments were performed at least three times and representative result was presented.
    Gapdh Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti gapdh
    Oligodendrocyte-specific deletion of Npc1 leads to blockade of oligodendrocyte maturation. (A) <t>NG2</t> (to mark OPCs) and CC1 (to mark mature oligodendrocytes) staining in the cortex and corpus callosum of P16 Npc1 flox/− , CNP Cre/+ and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (B) Western blot of NG2 expression levels from cerebral cortex homogenates of P16 Npc1 flox/− , CNP Cre/+ mice and controls. <t>GAPDH</t> controls for loading. (C) Quantification of CC1 + cell number in the corpus callosum of P16 Npc1 flox/− , CNP Cre/+ mice and controls. Data are mean +/− SD. * P
    Rabbit Anti Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam mouse anti gapdh
    Atpif 1 deficiency causes a decline in the retinal expression of <t>OPA1.</t> a , b Fluorescent IHC of whole-mount 72 hpf zebrafish, stained with anti-OPA1 antibody. Representative images ( a ) and quantification of OPA1 fluorescence intensity ( b ) are reported, showing a reduction in the OPA1 expression levels in the brain and retina of pnt tq209 larvae (OPA1 fluorescence (A.U.), Sb: 1.00 ± 0.01, pnt tq209 : 0.70 ± 0.09; results are presented as mean ± S.E.M. ( n = 3)). c , d Analysis of eye morphology in Sb and pnt tq209 zebrafish obtained through anti-acetylated α-tubulin IHC of whole-mount 72 hpf zebrafish. No major morphological differences were observed between normal and mutant larvae, as shown in the prototypical images ( c ) and relative quantification of eye size ( d ) (eye size (pixels), Sb: 2.58 ± 0.10, pnt tq209 : 2.53 ± 0.06; results are presented as mean ± S.E.M. ( n = 10)). e , f Quantification of OPA1 levels via western blotting analysis in 72 hpf wild type and pnt tq209 larvae. The representative membrane blotted for both OPA1 isoform ( e ) and the bar chart ( f ) show a significant decrease in the levels of the short isoform of the protein in pnt tq209 larvae. g – j (OPA1 band density relative to ACTB, OPA1 long isoform Sb: 1.00 ± 0.01, pnt tq209 :0.97 ± 0.01; OPA1 short isoform Sb: 1.00 ± 0.01, pnt tq209 :0.84 ± 0.01; results are presented as mean ± S.E.M. ( n = 3), Quantitative western blot analysis of OPA1 levels in the brain (frontal cortex/hippocampus) ( j , k ) and isolated optic nerve ( l , m ) of WT and Atpif1 −/− mice. Representative blots ( j , l ) and quantitated band densities relative to <t>GAPDH</t> ( k , m ) are reported. Significantly lower levels of short and long OPA1 isoforms expression were found in the brain and, specifically, in the optic nerve of mutant mice (OPA1 band density relative to GAPDH, OPA1 long isoform WT: 1.00 ± 0.01, Atpif1 −/− : 0.52 ± 0.01 in the frontal cortex/hippocampus; in the optic nerve, WT: 1.00 ± 0.05, Atpif1 −/− : 0.59 ± 0.09; in the frontal cortex/hippocampus OPA1 short isoform WT: 1.00 ± 0.01, Atpif 1−/− : 0.68 ± 0.01; in the optic nerve, WT: 1.00 ± 0.05, Atpif1 −/− : 0.548 ± 0.01; results are presented as mean ± S.E.M. ( n = 3). FB forebrain, HB hindbrain, MB midbrain, NR neural retina, L lens
    Mouse Anti Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A and B ) Local injection of emerin ( A ) or lamin A ( B ) lentiviruses (lenti) significantly increased the expression of emerin or lamin A/C in common carotid arteries the hypertensive rats. ( C ) Immunofluorescence staining against BrdU revealed that locally injected lentiviruses of emerin or lamin A remarkably decreased VSMC proliferation in the media of common carotid arteries of hypertensive rats. For Western blot, GAPDH was used for normalization. Values are expressed as mean ± SD. ** P

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

    Article Title: Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    doi: 10.1073/pnas.1604569113

    Figure Lengend Snippet: ( A and B ) Local injection of emerin ( A ) or lamin A ( B ) lentiviruses (lenti) significantly increased the expression of emerin or lamin A/C in common carotid arteries the hypertensive rats. ( C ) Immunofluorescence staining against BrdU revealed that locally injected lentiviruses of emerin or lamin A remarkably decreased VSMC proliferation in the media of common carotid arteries of hypertensive rats. For Western blot, GAPDH was used for normalization. Values are expressed as mean ± SD. ** P

    Article Snippet: The blots were incubated overnight at 4 °C with the following antibodies: lamin A/C (1:500) (Santa Cruz Technologies), emerin (1:500) (Abcam), lamin A (1:500) (Abcam), lamin C (1:500) (Abcam), and GAPDH (1:500) (Santa Cruz Technologies).

    Techniques: Injection, Expressing, Immunofluorescence, Staining, Western Blot

    Emerin and lamin A/C negatively regulate VSMC proliferation under different conditions (static control, 5% cyclic stretch, and 15% cyclic stretch). ( A ) Emerin and lamin A/C expression was increased after 5% cyclic stretch (CS) applied for 12 h and 24 h, compared with static control. GAPDH was used for normalization. Values are expressed as mean ± SD; * P

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

    Article Title: Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    doi: 10.1073/pnas.1604569113

    Figure Lengend Snippet: Emerin and lamin A/C negatively regulate VSMC proliferation under different conditions (static control, 5% cyclic stretch, and 15% cyclic stretch). ( A ) Emerin and lamin A/C expression was increased after 5% cyclic stretch (CS) applied for 12 h and 24 h, compared with static control. GAPDH was used for normalization. Values are expressed as mean ± SD; * P

    Article Snippet: The blots were incubated overnight at 4 °C with the following antibodies: lamin A/C (1:500) (Santa Cruz Technologies), emerin (1:500) (Abcam), lamin A (1:500) (Abcam), lamin C (1:500) (Abcam), and GAPDH (1:500) (Santa Cruz Technologies).

    Techniques: Expressing

    MG-132(R), a proteasome inhibitor, increased expression of emerin and lamin A/C in VSMCs subjected to 15% cyclic stretch for 24 h. GAPDH was used for normalization. Values are expressed as mean ± SD. * P

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

    Article Title: Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    doi: 10.1073/pnas.1604569113

    Figure Lengend Snippet: MG-132(R), a proteasome inhibitor, increased expression of emerin and lamin A/C in VSMCs subjected to 15% cyclic stretch for 24 h. GAPDH was used for normalization. Values are expressed as mean ± SD. * P

    Article Snippet: The blots were incubated overnight at 4 °C with the following antibodies: lamin A/C (1:500) (Santa Cruz Technologies), emerin (1:500) (Abcam), lamin A (1:500) (Abcam), lamin C (1:500) (Abcam), and GAPDH (1:500) (Santa Cruz Technologies).

    Techniques: Expressing

    Cyclic stretch (CS) modulated the expression of NE proteins emerin and lamin A/C, which participate in the stretch-induced proliferation of VSMCs in vitro. ( A ) A 15% cyclic stretch increased VSMC proliferation in comparison with a 5% cyclic stretch. ( B ) A 15% cyclic stretch decreased the expression of emerin and lamin A/C in comparison with a 5% cyclic stretch. ( C ) Under static conditions, target siRNA transfection significantly decreased and plasmid transfection significantly increased the expression of emerin. ( D ) Under static conditions, target siRNA transfection significantly decreased and plasmid transfections significantly increased the expression of lamin A/C. ( E ) Under static conditions, specific RNAi of both emerin and lamin A/C increased VSMC proliferation. ( F ) Up-regulated expression of emerin and lamin A/C decreased VSMC proliferation. For Western blots, GAPDH was used for normalization. Values are expressed as mean ± SD. * P

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

    Article Title: Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    doi: 10.1073/pnas.1604569113

    Figure Lengend Snippet: Cyclic stretch (CS) modulated the expression of NE proteins emerin and lamin A/C, which participate in the stretch-induced proliferation of VSMCs in vitro. ( A ) A 15% cyclic stretch increased VSMC proliferation in comparison with a 5% cyclic stretch. ( B ) A 15% cyclic stretch decreased the expression of emerin and lamin A/C in comparison with a 5% cyclic stretch. ( C ) Under static conditions, target siRNA transfection significantly decreased and plasmid transfection significantly increased the expression of emerin. ( D ) Under static conditions, target siRNA transfection significantly decreased and plasmid transfections significantly increased the expression of lamin A/C. ( E ) Under static conditions, specific RNAi of both emerin and lamin A/C increased VSMC proliferation. ( F ) Up-regulated expression of emerin and lamin A/C decreased VSMC proliferation. For Western blots, GAPDH was used for normalization. Values are expressed as mean ± SD. * P

    Article Snippet: The blots were incubated overnight at 4 °C with the following antibodies: lamin A/C (1:500) (Santa Cruz Technologies), emerin (1:500) (Abcam), lamin A (1:500) (Abcam), lamin C (1:500) (Abcam), and GAPDH (1:500) (Santa Cruz Technologies).

    Techniques: Expressing, In Vitro, Transfection, Plasmid Preparation, Western Blot

    In situ proliferation and expression of emerin and lamin A/C in the common carotid artery of hypertensive rats and sham-treated controls. ( A ) Immunofluorescence staining against BrdU revealed that after 1 wk of abdominal aorta coarctation, VSMC proliferation in the media of common carotid arteries increased markedly. ( B ) The expression of emerin and of lamin A/C was repressed significantly in the common carotid arteries of the aorta-coarctation–induced hypertensive rats. GAPDH was used for normalization. Values are expressed as mean ± SD. * P

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

    Article Title: Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    doi: 10.1073/pnas.1604569113

    Figure Lengend Snippet: In situ proliferation and expression of emerin and lamin A/C in the common carotid artery of hypertensive rats and sham-treated controls. ( A ) Immunofluorescence staining against BrdU revealed that after 1 wk of abdominal aorta coarctation, VSMC proliferation in the media of common carotid arteries increased markedly. ( B ) The expression of emerin and of lamin A/C was repressed significantly in the common carotid arteries of the aorta-coarctation–induced hypertensive rats. GAPDH was used for normalization. Values are expressed as mean ± SD. * P

    Article Snippet: The blots were incubated overnight at 4 °C with the following antibodies: lamin A/C (1:500) (Santa Cruz Technologies), emerin (1:500) (Abcam), lamin A (1:500) (Abcam), lamin C (1:500) (Abcam), and GAPDH (1:500) (Santa Cruz Technologies).

    Techniques: In Situ, Expressing, Immunofluorescence, Staining

    The expressions of emerin, lamin A, and lamin C are correlated with one other. ( A ) The siRNA (CAGG UCUG AAGC CAAA GAAT T) targets on LMNA , which is the template of both lamin A and lamin C. ( B ) Transfection of target siRNA to lamin A/C modulated the expression of both lamin A and lamin C. GAPDH was used for normalization. Values are expressed as mean ± SD. * P

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

    Article Title: Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    doi: 10.1073/pnas.1604569113

    Figure Lengend Snippet: The expressions of emerin, lamin A, and lamin C are correlated with one other. ( A ) The siRNA (CAGG UCUG AAGC CAAA GAAT T) targets on LMNA , which is the template of both lamin A and lamin C. ( B ) Transfection of target siRNA to lamin A/C modulated the expression of both lamin A and lamin C. GAPDH was used for normalization. Values are expressed as mean ± SD. * P

    Article Snippet: The blots were incubated overnight at 4 °C with the following antibodies: lamin A/C (1:500) (Santa Cruz Technologies), emerin (1:500) (Abcam), lamin A (1:500) (Abcam), lamin C (1:500) (Abcam), and GAPDH (1:500) (Santa Cruz Technologies).

    Techniques: Transfection, Expressing

    Cyclic stretch modulates the proliferation of VSMCs via NE proteins in vitro. ( A ) The expression of emerin or lamin A/C was repressed by target siRNA transfection during the application of 5% cyclic stretch. ( B ) Emerin- or lamin A/C-target siRNA transfection increased the proliferation of VSMCs. ( C ) The expression of emerin and of lamin A/C was increased by plasmid transfection. ( D ) Transfection with plasmid overexpressing emerin and lamin A decreased VSMC proliferation under 15% cyclic stretch. For Western blots, GAPDH was used for normalization. Values are expressed as mean ± SD. * P

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

    Article Title: Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    doi: 10.1073/pnas.1604569113

    Figure Lengend Snippet: Cyclic stretch modulates the proliferation of VSMCs via NE proteins in vitro. ( A ) The expression of emerin or lamin A/C was repressed by target siRNA transfection during the application of 5% cyclic stretch. ( B ) Emerin- or lamin A/C-target siRNA transfection increased the proliferation of VSMCs. ( C ) The expression of emerin and of lamin A/C was increased by plasmid transfection. ( D ) Transfection with plasmid overexpressing emerin and lamin A decreased VSMC proliferation under 15% cyclic stretch. For Western blots, GAPDH was used for normalization. Values are expressed as mean ± SD. * P

    Article Snippet: The blots were incubated overnight at 4 °C with the following antibodies: lamin A/C (1:500) (Santa Cruz Technologies), emerin (1:500) (Abcam), lamin A (1:500) (Abcam), lamin C (1:500) (Abcam), and GAPDH (1:500) (Santa Cruz Technologies).

    Techniques: In Vitro, Expressing, Transfection, Plasmid Preparation, Western Blot

    PKCε protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and GAPDH. HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: PKCε protein levels in HeLaPKCεA/E and glioblastoma cell lines. Protein extracts from HeLaPKCεA/E (−Dox), HeLaPKCεA/E (+Dox), U-118 MG, U-138 MG, T98G, LN18 cell lines were subjected to Western blot analysis using antibodies against PKCε and GAPDH. HeLaPKCεA/E cells with doxycycline-induced expression of PKCε (+Dox) and without doxycycline treatment (−Dox) served as a reference cell lines with low and high expression of PKCε, respectively. The average relative absorbance of three independent experiments is presented in a bar graph

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Western Blot, Expressing

    Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Activation Assay, Transfection, Construct

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection, Microscopy, Expressing

    Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: The following antibodies were used for detection: anti-PKCε, anti-BECN1, anti-Bcl-2, anti-FAK, anti-pFAK (Tyr-397), anti-pFAK (Tyr 576/577), anti-GAPDH, anti-rabbit IgG-HRP (all from Santa Cruz Biotechnology, CA, USA), anti-MAPLC3β (Sigma, St Louis, MO, USA), anti-ATG5, anti-mTOR, anti-SQSTM1/p62, anti-Akt and anti-pAkt (Ser473) (from Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection

    CERS6 binds to intracellular domains of CD95/Fas. a Constructs (pCMV6-mycDDK or pCMV6-AC-HA) encoding a human wild-type CERS6 tagged with mycDDK at COOH terminus, FAS full-length and FAS mutants tagged with HA epitopes. HD homeobox (DNA binding) domain, TLC TRAM/LAG/CLN8 homology domain, CRD cysteine-rich domain, TD transmembrane domain, DD death domain. b Co-immunoprecipitation of full-length CERS6 and Fas and Fas mutants to detect direct binding. c Co-immunoprecipitation of full-length CERS6 and Fas/Fas mutants in cellular fractions (Cyt cytosolic fraction that includes ER and other organelles, PM plasma membrane). GAPDH (right) is used as a marker for cytosolic fraction while E-cadherin is the marker for plasma membrane

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: CERS6 binds to intracellular domains of CD95/Fas. a Constructs (pCMV6-mycDDK or pCMV6-AC-HA) encoding a human wild-type CERS6 tagged with mycDDK at COOH terminus, FAS full-length and FAS mutants tagged with HA epitopes. HD homeobox (DNA binding) domain, TLC TRAM/LAG/CLN8 homology domain, CRD cysteine-rich domain, TD transmembrane domain, DD death domain. b Co-immunoprecipitation of full-length CERS6 and Fas and Fas mutants to detect direct binding. c Co-immunoprecipitation of full-length CERS6 and Fas/Fas mutants in cellular fractions (Cyt cytosolic fraction that includes ER and other organelles, PM plasma membrane). GAPDH (right) is used as a marker for cytosolic fraction while E-cadherin is the marker for plasma membrane

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Construct, Binding Assay, Thin Layer Chromatography, Immunoprecipitation, Marker

    CERS6 is overexpressed in ALL. a Biosynthesis and metabolism of ceramides. Ceramides are generated either de novo or via the salvage pathway. They are metabolized to sphingosine or may serve as substrates for the synthesis of glucosyl ceramides, lactosyl ceramides or sphingomyelins. Sphingomyelins degrade to ceramides via the sphingomyelinase pathway and are synthesized from ceramides via sphingomyelin synthase. b mRNA expression of the six ceramide synthase isoforms in the NCI PPTP panel of 23 cell lines comprising six different pediatric cancers. RBD rhabdomyosarcoma, BT brain tumor, EFT Ewing’s family of tumors, NB neuroblastoma, ALL acute lymphoblastic leukemia, LYM lymphoma. The mRNA expression was determined within the NCI Pediatric Preclinical Testing Program. c CERS6 protein expression in acute lymphoblastic leukemia (ALL) cell lines in comparison to peripheral blood mononuclear cells (PBMCs) and T lymphocytes obtained from blood of healthy human volunteers. GAPDH was used as a loading control. d C 16 -Ceramides in T-cell ALL cells in comparison to PBMCs and T lymphocytes. Ceramide levels were quantitated by HPLC/MS/MS (PBMC: 0.28 ± 0.04, n = 18; T lymphocytes: 0.36 ± 0.01, n = 3; T-ALL: 0.54 ± 0.09, n = 9). e CERS6 protein expression in T lymphocytes isolated from primary lymphoid malignancy samples in comparison to normal T lymphocytes *** p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: CERS6 is overexpressed in ALL. a Biosynthesis and metabolism of ceramides. Ceramides are generated either de novo or via the salvage pathway. They are metabolized to sphingosine or may serve as substrates for the synthesis of glucosyl ceramides, lactosyl ceramides or sphingomyelins. Sphingomyelins degrade to ceramides via the sphingomyelinase pathway and are synthesized from ceramides via sphingomyelin synthase. b mRNA expression of the six ceramide synthase isoforms in the NCI PPTP panel of 23 cell lines comprising six different pediatric cancers. RBD rhabdomyosarcoma, BT brain tumor, EFT Ewing’s family of tumors, NB neuroblastoma, ALL acute lymphoblastic leukemia, LYM lymphoma. The mRNA expression was determined within the NCI Pediatric Preclinical Testing Program. c CERS6 protein expression in acute lymphoblastic leukemia (ALL) cell lines in comparison to peripheral blood mononuclear cells (PBMCs) and T lymphocytes obtained from blood of healthy human volunteers. GAPDH was used as a loading control. d C 16 -Ceramides in T-cell ALL cells in comparison to PBMCs and T lymphocytes. Ceramide levels were quantitated by HPLC/MS/MS (PBMC: 0.28 ± 0.04, n = 18; T lymphocytes: 0.36 ± 0.01, n = 3; T-ALL: 0.54 ± 0.09, n = 9). e CERS6 protein expression in T lymphocytes isolated from primary lymphoid malignancy samples in comparison to normal T lymphocytes *** p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Generated, Synthesized, Expressing, High Performance Liquid Chromatography, Mass Spectrometry, Isolation

    ALL cells are sensitized to ABT-737 upon CERS6 knockdown. a CERS6 protein levels in CCRF-CEM and MOLT-4 cells after stably knocking down CERS6 using shRNA (NT-shRNA non-targeted shRNA, sh CERS6 -Mixed shRNA against CERS6 and selected with puromycin, sh CERS6 -Single shRNA against CERS6 and selected with puromycin followed by repopulation from a single cell). GAPDH was used as a loading control. b C 16 -Ceramide levels (pmole/nmole of inorganic phosphate) in ALL cells after CERS6 knockdown. Ceramide levels were quantitated by HPLC/MS/MS (1.5 ± 0.1 vs 7.3 ± 0.9 pmole/nmole Pi for CCRF-CEM and 7.4 ± 0.4 vs 10.9 ± 0.3 pmole/nmole Pi for MOLT-4; n = 3). c Knockdown of CERS6 in CCRF-CEM and MOLT-4 sensitized the cells to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 100 nM of ABT-737 in both cell lines (5.2 ± 2.7% vs 86.6 ± 4.6% survival at 100 nM ABT-737 for CCRF-CEM and 26.1 ± 3.8% vs 73.6 ± 4.6% survival at 100 nM ABT-737 for MOLT-4; n = 6). d Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (1 µM for 16 h) in CCRF-CEM or MOLT-4 cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA (98.0 ± 0.4% vs 38.0 ± 0.4% for CCRF-CEM and 90.8 ± 0.3% vs 72.1 ± 0.3% for MOLT-4; n = 3). e CERS6 knockdown in CCRF-CEM or MOLT-4 cells show higher levels of cleaved PARP and cleaved caspase-3 on ABT-737 treatment (1 µM for 16 h). GAPDH was used as a loading control * p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: ALL cells are sensitized to ABT-737 upon CERS6 knockdown. a CERS6 protein levels in CCRF-CEM and MOLT-4 cells after stably knocking down CERS6 using shRNA (NT-shRNA non-targeted shRNA, sh CERS6 -Mixed shRNA against CERS6 and selected with puromycin, sh CERS6 -Single shRNA against CERS6 and selected with puromycin followed by repopulation from a single cell). GAPDH was used as a loading control. b C 16 -Ceramide levels (pmole/nmole of inorganic phosphate) in ALL cells after CERS6 knockdown. Ceramide levels were quantitated by HPLC/MS/MS (1.5 ± 0.1 vs 7.3 ± 0.9 pmole/nmole Pi for CCRF-CEM and 7.4 ± 0.4 vs 10.9 ± 0.3 pmole/nmole Pi for MOLT-4; n = 3). c Knockdown of CERS6 in CCRF-CEM and MOLT-4 sensitized the cells to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 100 nM of ABT-737 in both cell lines (5.2 ± 2.7% vs 86.6 ± 4.6% survival at 100 nM ABT-737 for CCRF-CEM and 26.1 ± 3.8% vs 73.6 ± 4.6% survival at 100 nM ABT-737 for MOLT-4; n = 6). d Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (1 µM for 16 h) in CCRF-CEM or MOLT-4 cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA (98.0 ± 0.4% vs 38.0 ± 0.4% for CCRF-CEM and 90.8 ± 0.3% vs 72.1 ± 0.3% for MOLT-4; n = 3). e CERS6 knockdown in CCRF-CEM or MOLT-4 cells show higher levels of cleaved PARP and cleaved caspase-3 on ABT-737 treatment (1 µM for 16 h). GAPDH was used as a loading control * p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Stable Transfection, shRNA, High Performance Liquid Chromatography, Mass Spectrometry, Concentration Assay, Apoptosis Assay, Flow Cytometry, Cytometry, Transduction

    CERS6 alters ALL cells sensitivity to ABT-737 via the extrinsic pathway of apoptosis which can be overcome by a caspase-8 inhibitor. a Higher caspase-8 activity seen with CCRF-CEM and MOLT-4 CERS6 knockdown cells in comparison to cells transduced with non-targeted shRNA upon ABT-737 treatment. GAPDH was used as a loading control. b Annexin-V apoptosis assay by flow cytometry showing the percentage of apoptotic cells in ABT-737-treated CERS6 knockdown cells (CCRF-CEM and MOLT-4), with or without caspase-8 inhibitor, Z-IETD (23.4 ± 1.7% vs 37.5 ± 1.5% for CCRF-CEM and 8.2 ± 0.1% vs 20.8 ± 0.3% for MOLT-4; n = 3). c ABT-737-treated CERS6 knockdown cells show decreased levels of cleaved Caspase-8, cleaved PARP and cleaved caspase-3 on addition of Z-IETD in comparison to ABT-737-treated CERS6 knockdown cells without Z-IETD. d Levels of soluble FasL released in cultured medium upon treatment of ABT-737 in CERS6 knockdown cells compared to cells transduced with non-targeted shRNA. e Left: Caspase-8 activity was increased in a dose-dependent manner upon treatment of varying concentrations of Fas ligand (FasL) in CCRF-CEM cells with CERS6 knockdown. GAPDH was used as a loading control. e Right: Annexin-V apoptosis assay by flow cytometry showing increase in apoptotic cells in CCRF-CEM cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA upon treatment of 1000 ng/ml of FasL (70.9 ± 0.6% vs 14.1 ± 0.1%; n = 3) ** p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: CERS6 alters ALL cells sensitivity to ABT-737 via the extrinsic pathway of apoptosis which can be overcome by a caspase-8 inhibitor. a Higher caspase-8 activity seen with CCRF-CEM and MOLT-4 CERS6 knockdown cells in comparison to cells transduced with non-targeted shRNA upon ABT-737 treatment. GAPDH was used as a loading control. b Annexin-V apoptosis assay by flow cytometry showing the percentage of apoptotic cells in ABT-737-treated CERS6 knockdown cells (CCRF-CEM and MOLT-4), with or without caspase-8 inhibitor, Z-IETD (23.4 ± 1.7% vs 37.5 ± 1.5% for CCRF-CEM and 8.2 ± 0.1% vs 20.8 ± 0.3% for MOLT-4; n = 3). c ABT-737-treated CERS6 knockdown cells show decreased levels of cleaved Caspase-8, cleaved PARP and cleaved caspase-3 on addition of Z-IETD in comparison to ABT-737-treated CERS6 knockdown cells without Z-IETD. d Levels of soluble FasL released in cultured medium upon treatment of ABT-737 in CERS6 knockdown cells compared to cells transduced with non-targeted shRNA. e Left: Caspase-8 activity was increased in a dose-dependent manner upon treatment of varying concentrations of Fas ligand (FasL) in CCRF-CEM cells with CERS6 knockdown. GAPDH was used as a loading control. e Right: Annexin-V apoptosis assay by flow cytometry showing increase in apoptotic cells in CCRF-CEM cells with CERS6 knockdown in comparison to cells transduced with NT-shRNA upon treatment of 1000 ng/ml of FasL (70.9 ± 0.6% vs 14.1 ± 0.1%; n = 3) ** p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Activity Assay, Transduction, shRNA, Apoptosis Assay, Flow Cytometry, Cytometry, Cell Culture

    ALL cells overexpressing CERS6 show resistance to ABT-737. a CERS6 levels in CCRF-CEM cells upon CERS6 overexpression (Control cells transduced with empty vector, CERS6 -Mixed CERS6 overexpressing cells and selected with puromycin, CERS6 -Single CERS6 overexpressing cells and selected with puromycin followed by repopulation from a single cell; 10.1 ± 0.5 vs 5.7 ± 0.1 pmole/nmole Pi; n = 3). b C 16 -Cer levels (pmole/nmole of inorganic phosphate) in CCRF-CEM cells after CERS6 overexpression. Ceramide levels were quantitated by HPLC/MS/MS. c CERS6 overexpression in CCRF-CEM cells rendered the cells resistant to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 300 nM of ABT-737.(39.5 ± 2.2% vs 2.3 ± 1.8% survival at 300 nM ABT-737; n = 6) ( d ) Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (2 µM for 16 h) in CERS6 overexpressing CCRF-CEM cells in comparison to cells transduced with empty vector (36.8 ± 3.8% vs 85.2 ± 0.6%; n = 3). e CERS6 overexpressing CCRF-CEM cells show lower levels of cleaved PARP and cleaved Caspase-3 on ABT-737 treatment (2 µM for 16 h) in comparison to control cells. The far right lane (sh CERS6 -Single) is a sample from ABT-737-treated CERS6 knockdown CCRF-CEM cells (from Fig. 2e ) and represents positive control for cleaved PARP and cleaved caspase-3. GAPDH was used as a loading control * p

    Journal: Cell Death & Disease

    Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41419-018-0964-4

    Figure Lengend Snippet: ALL cells overexpressing CERS6 show resistance to ABT-737. a CERS6 levels in CCRF-CEM cells upon CERS6 overexpression (Control cells transduced with empty vector, CERS6 -Mixed CERS6 overexpressing cells and selected with puromycin, CERS6 -Single CERS6 overexpressing cells and selected with puromycin followed by repopulation from a single cell; 10.1 ± 0.5 vs 5.7 ± 0.1 pmole/nmole Pi; n = 3). b C 16 -Cer levels (pmole/nmole of inorganic phosphate) in CCRF-CEM cells after CERS6 overexpression. Ceramide levels were quantitated by HPLC/MS/MS. c CERS6 overexpression in CCRF-CEM cells rendered the cells resistant to ABT-737. Dose response curves showing concentration of ABT-737 on x -axis and survival fraction on y -axis on a log 10 scale. Bar graphs depict survival fractions at 300 nM of ABT-737.(39.5 ± 2.2% vs 2.3 ± 1.8% survival at 300 nM ABT-737; n = 6) ( d ) Annexin-V apoptosis assay by flow cytometry showing the percentage of live and apoptotic cells upon ABT-737 treatment (2 µM for 16 h) in CERS6 overexpressing CCRF-CEM cells in comparison to cells transduced with empty vector (36.8 ± 3.8% vs 85.2 ± 0.6%; n = 3). e CERS6 overexpressing CCRF-CEM cells show lower levels of cleaved PARP and cleaved Caspase-3 on ABT-737 treatment (2 µM for 16 h) in comparison to control cells. The far right lane (sh CERS6 -Single) is a sample from ABT-737-treated CERS6 knockdown CCRF-CEM cells (from Fig. 2e ) and represents positive control for cleaved PARP and cleaved caspase-3. GAPDH was used as a loading control * p

    Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).

    Techniques: Over Expression, Transduction, Plasmid Preparation, High Performance Liquid Chromatography, Mass Spectrometry, Concentration Assay, Apoptosis Assay, Flow Cytometry, Cytometry, Positive Control

    Cyclin B1-Cdc2 complex maintains G2/M arrest (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Cyclin B1-Cdc2 complex maintains G2/M arrest (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Cell Cycle Assay, Staining, Cell Counting, Irradiation, Incubation, MTT Assay, Western Blot

    Irradiated meningioma form aggressive tumors while uPAR knock down induces sustained G2/M arrest in vivo (A-B) IOMM Lee and CH 157 MN luciferase expressing stable cells (1×10 5 ) were implanted into nude mice (4 to 6 weeks old). The first group was treated with irradiated (7 Gy) cells. The second group was infused with irradiated (7 Gy) cells that were allowed to recover. The third group was infused with non-irradiated cells. Tumor progression and morphological and behavioral patterns were followed daily for two weeks with an in vivo imaging system. (C) Representative photmicrograph (40X)of Immunohistochemistry for Cyclin B1 in the brain sections of animals implanted with transfected IOMM Lee cells in combination with radiation treatment (IR; 7Gy) as indicated (n=5). (D) H E staining of brain sections for the visualization of tumor formation. (E) Representative image of RT-PCR analysis for Cyclin B1 in the brain tissues of different treatment groups. GAPDH served as loading control (n=3).

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Irradiated meningioma form aggressive tumors while uPAR knock down induces sustained G2/M arrest in vivo (A-B) IOMM Lee and CH 157 MN luciferase expressing stable cells (1×10 5 ) were implanted into nude mice (4 to 6 weeks old). The first group was treated with irradiated (7 Gy) cells. The second group was infused with irradiated (7 Gy) cells that were allowed to recover. The third group was infused with non-irradiated cells. Tumor progression and morphological and behavioral patterns were followed daily for two weeks with an in vivo imaging system. (C) Representative photmicrograph (40X)of Immunohistochemistry for Cyclin B1 in the brain sections of animals implanted with transfected IOMM Lee cells in combination with radiation treatment (IR; 7Gy) as indicated (n=5). (D) H E staining of brain sections for the visualization of tumor formation. (E) Representative image of RT-PCR analysis for Cyclin B1 in the brain tissues of different treatment groups. GAPDH served as loading control (n=3).

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Irradiation, In Vivo, Luciferase, Expressing, Mouse Assay, In Vivo Imaging, Immunohistochemistry, Transfection, Staining, Reverse Transcription Polymerase Chain Reaction

    Expression of cell cycle-related proteins in irradiated cells (A) Western blot analysis of Chk2, phospho-Chk2, p53, Cdc25C, phospho-Cdc25C (Ser216), cyclin B1 and phospho-Cdc2 (Thr 14/Tyr15) in IOMM Lee, CH 157 MN and SF3061 cells at 24 hrs post-irradiation. GAPDH served as a loading control. (B) ImageJ quantification of the molecules in arbitrary units; each value is representative of three independent experiments. (C) Western blot analysis of cyclin B1 and pCdc2 (Thr 14/Tyr 15) at 8 and 48 hrs post-irradiation. GAPDH served as a loading control.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Expression of cell cycle-related proteins in irradiated cells (A) Western blot analysis of Chk2, phospho-Chk2, p53, Cdc25C, phospho-Cdc25C (Ser216), cyclin B1 and phospho-Cdc2 (Thr 14/Tyr15) in IOMM Lee, CH 157 MN and SF3061 cells at 24 hrs post-irradiation. GAPDH served as a loading control. (B) ImageJ quantification of the molecules in arbitrary units; each value is representative of three independent experiments. (C) Western blot analysis of cyclin B1 and pCdc2 (Thr 14/Tyr 15) at 8 and 48 hrs post-irradiation. GAPDH served as a loading control.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Irradiation, Western Blot

    p53 acts as downstream effector to Chk2 in IOMM Lee cells (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Next, cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is a representative of three independent experiments. (D) ImageJ quantification of cyclin B1 and pCdc2 in the inhibitor-treated cells.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: p53 acts as downstream effector to Chk2 in IOMM Lee cells (A) Cell cycle analysis of IOMM Lee, CH 157 MN and SF3061 cells treated with 10 μM inhibitor for 1 hr and/or 7 Gy radiation, stained with propidium iodide, and measured 24 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) IOMM Lee, CH 157 MN and SF3061 cells (1×10 5 ) were seeded in 6-well plates and irradiated. Next, cells were trypsinized and seeded at 1×10 4 cells per well in 96-well plates. After the indicated hours of incubation, MTT reagent was added, followed by another 4 hrs of incubation and the addition of acid-isopropanol. Absorbance was measured at 550 nm and the values were quantified. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 in the inhibitor-treated cells. GAPDH served as a loading control. Each blot is a representative of three independent experiments. (D) ImageJ quantification of cyclin B1 and pCdc2 in the inhibitor-treated cells.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Cell Cycle Assay, Staining, Cell Counting, Irradiation, Incubation, MTT Assay, Western Blot

    Meningioma cells recover from radiation induced cell cycle arrest and uPAR knock down induces cell death (A) Cell cycle analysis of IOMM Lee and CH 157 MN cells treated with 7 Gy radiation, stained with propidium iodide, and measured 72 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) Quantification of distribution of cells in the G0/G1 and G2/M phases of the cell cycle. Data presented are means ± SEM calculated from three independent experiments. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 at 72 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments. (D) Cell cycle analysis of IOMM Lee and CH 157 MN cells transfected with Scrambled vector (SV), ShuPAR, irradiated (7Gy) stained with propidium iodide after 24 hrs treatment as indicated. Percentage of cells in SubG0/G1 population in each treatment group is plotted. Data presented are means ± SEM calculated from three independent experiments. E) IOMM Lee and CH 157 MN cells were transfected either with SV or ShuPAR expressing plasmids alone or in combination with radiation. After 6h radiation treatment cells were transferred into 96 well plates and MTT assay was performed at indicated period of times. Data presented are means ± SEM calculated from three independent experiments. F) Western blot analysis in transfected cells for cyclin B1 and pCdc2 at 24 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Journal: Cancer letters

    Article Title: Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells

    doi: 10.1016/j.canlet.2011.08.022

    Figure Lengend Snippet: Meningioma cells recover from radiation induced cell cycle arrest and uPAR knock down induces cell death (A) Cell cycle analysis of IOMM Lee and CH 157 MN cells treated with 7 Gy radiation, stained with propidium iodide, and measured 72 hrs after treatment. The y axis denotes cell count and the x axis represents DNA content. The percentages of cells out of 10,000 events were calculated without gating. (B) Quantification of distribution of cells in the G0/G1 and G2/M phases of the cell cycle. Data presented are means ± SEM calculated from three independent experiments. (C) Western blot analysis of pChk2, cyclin B1 and pCdc2 at 72 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments. (D) Cell cycle analysis of IOMM Lee and CH 157 MN cells transfected with Scrambled vector (SV), ShuPAR, irradiated (7Gy) stained with propidium iodide after 24 hrs treatment as indicated. Percentage of cells in SubG0/G1 population in each treatment group is plotted. Data presented are means ± SEM calculated from three independent experiments. E) IOMM Lee and CH 157 MN cells were transfected either with SV or ShuPAR expressing plasmids alone or in combination with radiation. After 6h radiation treatment cells were transferred into 96 well plates and MTT assay was performed at indicated period of times. Data presented are means ± SEM calculated from three independent experiments. F) Western blot analysis in transfected cells for cyclin B1 and pCdc2 at 24 hrs after irradiation. GAPDH served as a loading control. Each blot is representative of three independent experiments.

    Article Snippet: Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C, treated with NSC 109555 ditosylate or Nutlin or Aloisine (Santa Cruz Biotechnology, Santa Cruz, CA), and incubated for the indicated period of time in complete medium. pChk2 (Thr 68), Chk2, p53, cyclin B1, Cdc25C, pCdc25c (Ser 216), pCdc2 (Thr 14/Tyr15) and GAPDH antibodies were obtained from (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Cell Cycle Assay, Staining, Cell Counting, Western Blot, Irradiation, Transfection, Plasmid Preparation, Expressing, MTT Assay

    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing

    Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing

    TCR signaling induces RORγt phosphorylation and subsequent AhR-RORγt interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and GAPDH proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.

    Journal: Science Advances

    Article Title: GLK-IKKβ signaling induces dimerization and translocation of the AhR-RORγt complex in IL-17A induction and autoimmune disease

    doi: 10.1126/sciadv.aat5401

    Figure Lengend Snippet: TCR signaling induces RORγt phosphorylation and subsequent AhR-RORγt interaction. ( A ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, p-IKKβ (Ser 180/181 ), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( B ) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( C ) Immunoblotting analysis of p-RORγt (Ser 489 ), RORγt, and IKKβ in primary splenic T cells of IKKβ f/f or CD4-Cre;IKKβ f/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). ( D ) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser 489 -phosphorylated RORγt (right) in primary T cells of IKKβ f/f or IKKβ f/f ;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. ( E and F ) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβ f/f or IKKβ f/f ;CD4-Cre mice (E), as well as RORγt f/f or RORγt f/f ;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. ( G ) Immunoblotting of RORγt and GAPDH proteins from primary splenic T cells of RORγt f/f or RORγt f/f ;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. ( H ) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser 36 phosphorylation through PKCθ and also induces RORγt Ser 489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser 489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.

    Article Snippet: Anti-AhR (clone RPT9), anti–p-IKKβ (Ser180/181 ; #ab55341), and anti-GAPDH (clone mAbcam 9484, catalog #ab9482) antibodies were purchased from Abcam.

    Techniques: Mouse Assay, Confocal Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Over Expression, Binding Assay, Activity Assay, Activation Assay

    Western blot analysis of transforming growth factor beta (TGF‐β) and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase (GAPDH) antibodies. (b) Arbitrary values expressed as mean and SD. * P

    Journal: JGH Open: An Open Access Journal of Gastroenterology and Hepatology

    Article Title: Antifibrogenic effect of melatonin in rats with experimental liver cirrhosis induced by carbon tetrachloride

    doi: 10.1002/jgh3.12055

    Figure Lengend Snippet: Western blot analysis of transforming growth factor beta (TGF‐β) and alpha‐smooth muscle actin (α‐SMA). (a) Cytoplasmic fractions were analyzed by WB with TGF‐β and α‐SMA and glyceraldehyde phosphate dehydrogenase (GAPDH) antibodies. (b) Arbitrary values expressed as mean and SD. * P

    Article Snippet: The anti‐β‐actin (A5060/42 kDa) and GAPDH (G9545/37 kDa) antibody (Sigma Aldrich) was at 1:2000 dilution with TTBS in 5% nonfat dry milk.

    Techniques: Western Blot

    Inhibition of clathrin-mediated endocytosis does not affect mitochondrial morphology a , Representative images of TOM20 immunofluorescence in n = 50, 50, 52 COS-7 cells transfected with scrambled control, AP-2, or Dyn2 siRNA. Scale bars = 10 μm. b , Immuno-blots with antibodies against AP-2, Dyn-2, and GAPDH in siRNA-treated cells. c , The effect on mitochondrial morphology was quantitated within a 230 μm 2 region of interest (ROI) for mean area per mitochondria (left graph), and mean mitochondria per ROI (right graph). As in , Dyn2–depleted cells had larger mitochondria and less mitochondrion per ROI compared to control cells; however, AP-2 depleted cells displayed mitochondrial morphology that was qualitatively and quantitatively similar to control cells. These data were obtained from three biological replicate experiments. Error bars represent the s.e.m. *p

    Journal: Nature

    Article Title: Multiple Dynamin family members collaborate to drive mitochondrial division

    doi: 10.1038/nature20555

    Figure Lengend Snippet: Inhibition of clathrin-mediated endocytosis does not affect mitochondrial morphology a , Representative images of TOM20 immunofluorescence in n = 50, 50, 52 COS-7 cells transfected with scrambled control, AP-2, or Dyn2 siRNA. Scale bars = 10 μm. b , Immuno-blots with antibodies against AP-2, Dyn-2, and GAPDH in siRNA-treated cells. c , The effect on mitochondrial morphology was quantitated within a 230 μm 2 region of interest (ROI) for mean area per mitochondria (left graph), and mean mitochondria per ROI (right graph). As in , Dyn2–depleted cells had larger mitochondria and less mitochondrion per ROI compared to control cells; however, AP-2 depleted cells displayed mitochondrial morphology that was qualitatively and quantitatively similar to control cells. These data were obtained from three biological replicate experiments. Error bars represent the s.e.m. *p

    Article Snippet: Protein levels in whole-cell lysates and cell fractions were assayed by Western blot with polyclonal rabbit antibodies against Dyn2 (ab3457, Abcam), phosphoSerine637-Drp1 (4867S, Cell Signaling Tech), phosphoSerine616-Drp1 (3455S, Cell Signaling Tech), Mff (17090-1-AP, Protein Tech), Mfn2 clone 4H8 (WH0009927M3, Sigma-Aldrich), α-Tubulin (ab18251, Abcam), TOM20 (sc-11415, Santa Cruz Biotech), Bax (sc-493, Santa Cruz Biotech), GAPDH (G9545, Sigma-Aldrich), and the monoclonal mouse antibody against AP-1/2 (sc-17771, Santa Cruz Biotech.), Drp1 (ab56788, Abcam), Dynamin (610245, BD Biosciences), cytochrome C (sc-13560, Santa Cruz Biotech), Opa1 (612606, BD Biosciences).

    Techniques: Inhibition, Immunofluorescence, Transfection, Western Blot

    Kidney-replenishing herb promoted SOCS-3 protein expression in human first-trimester trophoblasts. Primary human trophoblasts were incubated for 4 h with 10% to 20% Kidney-replenishing herb. As shown in (a), (b) there is also an increase of SOCS-3 protein expression (0.5, 1, 2, and 4 h). Control cells were treated with serum in the same way without herb and detected a trace SOCS-3 expression at 2 h (c). The levels of GAPDH were examined as an internal control (d). The experiments were performed at least three times and representative result was presented.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Kidney-Replenishing Herb Induces SOCS-3 Expression via ERK/MAPK Pathway and Improves Growth of the First-Trimester Human Trophoblast Cells

    doi: 10.1155/2017/2473431

    Figure Lengend Snippet: Kidney-replenishing herb promoted SOCS-3 protein expression in human first-trimester trophoblasts. Primary human trophoblasts were incubated for 4 h with 10% to 20% Kidney-replenishing herb. As shown in (a), (b) there is also an increase of SOCS-3 protein expression (0.5, 1, 2, and 4 h). Control cells were treated with serum in the same way without herb and detected a trace SOCS-3 expression at 2 h (c). The levels of GAPDH were examined as an internal control (d). The experiments were performed at least three times and representative result was presented.

    Article Snippet: Rabbit anti-human SOCS-3 antibodies, mouse anti-phosphoERK monoclonal antibodies, rabbit anti-ERK monoclonal antibodies, and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Incubation

    Modulation of Kidney-replenishing herb on growth of the primary cultured human first-trimester trophoblast cells. To verify rapid activation of ERK1/2, cells were starved for 12 h, subjected to 10% or 20% of Kidney-replenishing herb treatments for 4 h. The phosphorylation of ERK1/2 was observed in response to Kidney-replenishing herb treatment ((a), (b)). The phosphorylation of ERK1/2 induced by 20% of control serum was also detected (c). The samples were controlled by immunoblot against ERK (d) and GAPDH (e). The primary cultured trophoblasts were starved with 1% FBS for 12 h and then treated with vehicle, 10% Kidney-replenishing herb, 20% Kidney-replenishing herb, 10% control serum, 20% control serum, and Kidney-replenishing herb combined with U0126 (30 mmol/l) or U0126 (30 mmol/l) alone, respectively, for 48 h. The percent of PCNA expression (f) or Annexin-V-FITC/PI staining (g) was observed by flow cytometry. ∗ p

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Kidney-Replenishing Herb Induces SOCS-3 Expression via ERK/MAPK Pathway and Improves Growth of the First-Trimester Human Trophoblast Cells

    doi: 10.1155/2017/2473431

    Figure Lengend Snippet: Modulation of Kidney-replenishing herb on growth of the primary cultured human first-trimester trophoblast cells. To verify rapid activation of ERK1/2, cells were starved for 12 h, subjected to 10% or 20% of Kidney-replenishing herb treatments for 4 h. The phosphorylation of ERK1/2 was observed in response to Kidney-replenishing herb treatment ((a), (b)). The phosphorylation of ERK1/2 induced by 20% of control serum was also detected (c). The samples were controlled by immunoblot against ERK (d) and GAPDH (e). The primary cultured trophoblasts were starved with 1% FBS for 12 h and then treated with vehicle, 10% Kidney-replenishing herb, 20% Kidney-replenishing herb, 10% control serum, 20% control serum, and Kidney-replenishing herb combined with U0126 (30 mmol/l) or U0126 (30 mmol/l) alone, respectively, for 48 h. The percent of PCNA expression (f) or Annexin-V-FITC/PI staining (g) was observed by flow cytometry. ∗ p

    Article Snippet: Rabbit anti-human SOCS-3 antibodies, mouse anti-phosphoERK monoclonal antibodies, rabbit anti-ERK monoclonal antibodies, and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Cell Culture, Activation Assay, Expressing, Staining, Flow Cytometry, Cytometry

    10% Kidney-replenishing herb-induced ERK1/2 phosphorylation was not affected by SOCS-3 interference in JAR cells. JAR cells were starved for 24 h, subjected to 10% Kidney-replenishing herb treatments for 8 h. The phosphorylation of ERK1/2 was observed in response to vehicle control (a) and 10% control serum (b), the protein levels of pERK1/2 was significantly increased after treatment with 10% Kidney-replenishing herb (c). The samples were controlled by immunoblot against ERK (d) and GAPDH (e).

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Kidney-Replenishing Herb Induces SOCS-3 Expression via ERK/MAPK Pathway and Improves Growth of the First-Trimester Human Trophoblast Cells

    doi: 10.1155/2017/2473431

    Figure Lengend Snippet: 10% Kidney-replenishing herb-induced ERK1/2 phosphorylation was not affected by SOCS-3 interference in JAR cells. JAR cells were starved for 24 h, subjected to 10% Kidney-replenishing herb treatments for 8 h. The phosphorylation of ERK1/2 was observed in response to vehicle control (a) and 10% control serum (b), the protein levels of pERK1/2 was significantly increased after treatment with 10% Kidney-replenishing herb (c). The samples were controlled by immunoblot against ERK (d) and GAPDH (e).

    Article Snippet: Rabbit anti-human SOCS-3 antibodies, mouse anti-phosphoERK monoclonal antibodies, rabbit anti-ERK monoclonal antibodies, and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques:

    Kidney-replenishing herb induces phosphorylation of ERK1/2 and increases SOCS-3 protein. JAR cells were serum-starved for 24 h and stimulated with 10% Kidney-replenishing herb for 8 h. Western blot analysis was performed using antibodies against pERK1/2, ERK1/2, SOCS-3, and GAPDH.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Kidney-Replenishing Herb Induces SOCS-3 Expression via ERK/MAPK Pathway and Improves Growth of the First-Trimester Human Trophoblast Cells

    doi: 10.1155/2017/2473431

    Figure Lengend Snippet: Kidney-replenishing herb induces phosphorylation of ERK1/2 and increases SOCS-3 protein. JAR cells were serum-starved for 24 h and stimulated with 10% Kidney-replenishing herb for 8 h. Western blot analysis was performed using antibodies against pERK1/2, ERK1/2, SOCS-3, and GAPDH.

    Article Snippet: Rabbit anti-human SOCS-3 antibodies, mouse anti-phosphoERK monoclonal antibodies, rabbit anti-ERK monoclonal antibodies, and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Western Blot

    RT-PCR analysis of SOCS-3 mRNA expression after transfected with Stealth siRNA at 48 h. M: marker; lane 1, 2: cells transfected with Stealth siRNA; lane 3: cells transfected negative control; and lanes 4: untransfected cells. GAPDH expression was used as an internal control. Molecular weight is shown on the left.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Kidney-Replenishing Herb Induces SOCS-3 Expression via ERK/MAPK Pathway and Improves Growth of the First-Trimester Human Trophoblast Cells

    doi: 10.1155/2017/2473431

    Figure Lengend Snippet: RT-PCR analysis of SOCS-3 mRNA expression after transfected with Stealth siRNA at 48 h. M: marker; lane 1, 2: cells transfected with Stealth siRNA; lane 3: cells transfected negative control; and lanes 4: untransfected cells. GAPDH expression was used as an internal control. Molecular weight is shown on the left.

    Article Snippet: Rabbit anti-human SOCS-3 antibodies, mouse anti-phosphoERK monoclonal antibodies, rabbit anti-ERK monoclonal antibodies, and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Marker, Negative Control, Molecular Weight

    Oligodendrocyte-specific deletion of Npc1 leads to blockade of oligodendrocyte maturation. (A) NG2 (to mark OPCs) and CC1 (to mark mature oligodendrocytes) staining in the cortex and corpus callosum of P16 Npc1 flox/− , CNP Cre/+ and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (B) Western blot of NG2 expression levels from cerebral cortex homogenates of P16 Npc1 flox/− , CNP Cre/+ mice and controls. GAPDH controls for loading. (C) Quantification of CC1 + cell number in the corpus callosum of P16 Npc1 flox/− , CNP Cre/+ mice and controls. Data are mean +/− SD. * P

    Journal: PLoS Genetics

    Article Title: Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

    doi: 10.1371/journal.pgen.1003462

    Figure Lengend Snippet: Oligodendrocyte-specific deletion of Npc1 leads to blockade of oligodendrocyte maturation. (A) NG2 (to mark OPCs) and CC1 (to mark mature oligodendrocytes) staining in the cortex and corpus callosum of P16 Npc1 flox/− , CNP Cre/+ and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (B) Western blot of NG2 expression levels from cerebral cortex homogenates of P16 Npc1 flox/− , CNP Cre/+ mice and controls. GAPDH controls for loading. (C) Quantification of CC1 + cell number in the corpus callosum of P16 Npc1 flox/− , CNP Cre/+ mice and controls. Data are mean +/− SD. * P

    Article Snippet: Antibodies used were rat anti-MBP (1∶2000, Abcam), mouse anti-CNP (1∶2000, Millipore), mouse anti-MAG (1∶5000, Millipore), mouse anti-Neurofilament 200 (1∶5000, Millipore), rabbit anti-NG2 (1∶1000, Millipore), rabbit anti-GAPDH (1∶5000, Santa Cruz), mouse anti-Cre (1∶1000, Millipore), rabbit anti-GFAP (1∶5000, Dako), mouse anti-PSA-NCAM (1∶1000, Millipore), goat anti-Jagged1(1∶1000, Santa Cruz) and rabbit anti-Lingo1 (1∶1000, Abcam).

    Techniques: Staining, Mouse Assay, Western Blot, Expressing

    Forebrain dysmyelination in mice following neuron-specific deletion of Npc1 . (A) Schematic of midline sagittal section of the mouse brain, with the area shown in panel B highlighted by the black rectangle. Illustration is from www.gensat.org . (B) (Top three panels) MBP immunofluorescence in forebrain sagittal sections of Npc1 flox/− , Syn1-Cre + and control mice at P16, and at 7 16 weeks. Bar, 500 µm. Area highlighted by white rectangle is enlarged in the inset in the upper right of each panel. (Bottom panel) FluoroMyelin staining of the corpus callosm of Npc1 flox/− , Syn1-Cre + and control mice at 16 weeks. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (C) Western blots of myelin-specific proteins and neurofilament protein (NF-200) from brainstem and cerebral cortex homogenates of P16 Npc1 flox/− , Syn1-Cre + mice and controls. GAPDH controls for loading. (D) MBP and neurofilament protein (NF) co-staining of P16 Npc1 flox/− , Syn1-Cre + and littermate control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (E) Electron microscopy of the corpus callosum of P16 Npc1 flox/− , Syn1-Cre and control mice. Bar, 500 nm.

    Journal: PLoS Genetics

    Article Title: Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

    doi: 10.1371/journal.pgen.1003462

    Figure Lengend Snippet: Forebrain dysmyelination in mice following neuron-specific deletion of Npc1 . (A) Schematic of midline sagittal section of the mouse brain, with the area shown in panel B highlighted by the black rectangle. Illustration is from www.gensat.org . (B) (Top three panels) MBP immunofluorescence in forebrain sagittal sections of Npc1 flox/− , Syn1-Cre + and control mice at P16, and at 7 16 weeks. Bar, 500 µm. Area highlighted by white rectangle is enlarged in the inset in the upper right of each panel. (Bottom panel) FluoroMyelin staining of the corpus callosm of Npc1 flox/− , Syn1-Cre + and control mice at 16 weeks. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (C) Western blots of myelin-specific proteins and neurofilament protein (NF-200) from brainstem and cerebral cortex homogenates of P16 Npc1 flox/− , Syn1-Cre + mice and controls. GAPDH controls for loading. (D) MBP and neurofilament protein (NF) co-staining of P16 Npc1 flox/− , Syn1-Cre + and littermate control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (E) Electron microscopy of the corpus callosum of P16 Npc1 flox/− , Syn1-Cre and control mice. Bar, 500 nm.

    Article Snippet: Antibodies used were rat anti-MBP (1∶2000, Abcam), mouse anti-CNP (1∶2000, Millipore), mouse anti-MAG (1∶5000, Millipore), mouse anti-Neurofilament 200 (1∶5000, Millipore), rabbit anti-NG2 (1∶1000, Millipore), rabbit anti-GAPDH (1∶5000, Santa Cruz), mouse anti-Cre (1∶1000, Millipore), rabbit anti-GFAP (1∶5000, Dako), mouse anti-PSA-NCAM (1∶1000, Millipore), goat anti-Jagged1(1∶1000, Santa Cruz) and rabbit anti-Lingo1 (1∶1000, Abcam).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Western Blot, Electron Microscopy

    The effect of timing of Npc1 deletion on CNS myelination. (A) MBP immunofluorescence in brain midline sagittal sections of 7-week-old WT (top), 7-week-old Npc1 Δ/− (middle), and 22-week-old Npc1 flox/− , Cre-ER TM + mice following tamoxifen injections at 6 weeks (bottom). Bar, 1 mm. (B) FluoroMyelin staining of forebrain regions of 20-week-old Npc1 flox/+ , Cre-ER TM + control following tamoxifen injections at 6 weeks (top), 7-week-old Npc1 Δ/− (middle), and 22-week-old Npc1 flox/− , Cre-ER TM + mice following tamoxifen injections at 6 weeks (bottom). Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 500 µm. (C, D) Western blots of CNP, MBP and neurofilament protein (NF-200) expression levels from cerebral cortex (C) and cerebellar (D) homogenates of 22-week-old Npc1 flox/− , Cre-ER TM + mice and their littermate controls following tamoxifen injections at 6 weeks. GAPDH controls for loading.

    Journal: PLoS Genetics

    Article Title: Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

    doi: 10.1371/journal.pgen.1003462

    Figure Lengend Snippet: The effect of timing of Npc1 deletion on CNS myelination. (A) MBP immunofluorescence in brain midline sagittal sections of 7-week-old WT (top), 7-week-old Npc1 Δ/− (middle), and 22-week-old Npc1 flox/− , Cre-ER TM + mice following tamoxifen injections at 6 weeks (bottom). Bar, 1 mm. (B) FluoroMyelin staining of forebrain regions of 20-week-old Npc1 flox/+ , Cre-ER TM + control following tamoxifen injections at 6 weeks (top), 7-week-old Npc1 Δ/− (middle), and 22-week-old Npc1 flox/− , Cre-ER TM + mice following tamoxifen injections at 6 weeks (bottom). Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 500 µm. (C, D) Western blots of CNP, MBP and neurofilament protein (NF-200) expression levels from cerebral cortex (C) and cerebellar (D) homogenates of 22-week-old Npc1 flox/− , Cre-ER TM + mice and their littermate controls following tamoxifen injections at 6 weeks. GAPDH controls for loading.

    Article Snippet: Antibodies used were rat anti-MBP (1∶2000, Abcam), mouse anti-CNP (1∶2000, Millipore), mouse anti-MAG (1∶5000, Millipore), mouse anti-Neurofilament 200 (1∶5000, Millipore), rabbit anti-NG2 (1∶1000, Millipore), rabbit anti-GAPDH (1∶5000, Santa Cruz), mouse anti-Cre (1∶1000, Millipore), rabbit anti-GFAP (1∶5000, Dako), mouse anti-PSA-NCAM (1∶1000, Millipore), goat anti-Jagged1(1∶1000, Santa Cruz) and rabbit anti-Lingo1 (1∶1000, Abcam).

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

    Neuron-specific deletion of Npc1 leads to blockade of oligodendrocyte maturation. (A) NG2 (to mark OPCs) and CC1 (to mark mature oligodendrocytes) staining in the corpus callosum of P16 Npc1 flox/− , Syn1-Cre + and control mice. Bar, 200 µm. (B) Western blot of NG2 expression levels from cerebral cortex homogenates of P16 Npc1 flox/− , Syn1-Cre + mice and controls. GAPDH controls for loading. (C) Quantification of CC1 + cell number in the corpus callosum of P16 Npc1 flox/− , Syn1-Cre + mice and controls. Data are mean +/− SD. *** P

    Journal: PLoS Genetics

    Article Title: Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

    doi: 10.1371/journal.pgen.1003462

    Figure Lengend Snippet: Neuron-specific deletion of Npc1 leads to blockade of oligodendrocyte maturation. (A) NG2 (to mark OPCs) and CC1 (to mark mature oligodendrocytes) staining in the corpus callosum of P16 Npc1 flox/− , Syn1-Cre + and control mice. Bar, 200 µm. (B) Western blot of NG2 expression levels from cerebral cortex homogenates of P16 Npc1 flox/− , Syn1-Cre + mice and controls. GAPDH controls for loading. (C) Quantification of CC1 + cell number in the corpus callosum of P16 Npc1 flox/− , Syn1-Cre + mice and controls. Data are mean +/− SD. *** P

    Article Snippet: Antibodies used were rat anti-MBP (1∶2000, Abcam), mouse anti-CNP (1∶2000, Millipore), mouse anti-MAG (1∶5000, Millipore), mouse anti-Neurofilament 200 (1∶5000, Millipore), rabbit anti-NG2 (1∶1000, Millipore), rabbit anti-GAPDH (1∶5000, Santa Cruz), mouse anti-Cre (1∶1000, Millipore), rabbit anti-GFAP (1∶5000, Dako), mouse anti-PSA-NCAM (1∶1000, Millipore), goat anti-Jagged1(1∶1000, Santa Cruz) and rabbit anti-Lingo1 (1∶1000, Abcam).

    Techniques: Staining, Mouse Assay, Western Blot, Expressing

    Forebrain dysmyelination in mice with oligodendrocyte-specific deletion of Npc1 . (A) (Top panel) FluoroMyelin staining of the corpus callosum of Npc1 flox/− , CNP Cre/+ and control mice at P16. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (Bottom two panels) MBP immunofluorescence in forebrain sagittal sections of Npc1 flox/− , CNP Cre/+ mice and controls at P16 and 7 weeks. Bar, 500 µm. Area highlighted by white rectangle is enlarged in the inset in the upper right of each panel. (B) Western blots of myelin-specific proteins and neurofilament protein (NF-200) from brainstem and cerebral cortex homogenates of P16 Npc1 flox/− , CNP Cre/+ mice and controls. GAPDH controls for loading. (C) MBP and neurofilament protein (NF) co-staining in the corpus callosum of P16 Npc1 flox/− , CNP Cre/+ and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (D) Electron microscopy of the corpus callosum of a P16 Npc1 flox/− , CNP Cre/+ and control mice. Bar, 50 nm.

    Journal: PLoS Genetics

    Article Title: Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

    doi: 10.1371/journal.pgen.1003462

    Figure Lengend Snippet: Forebrain dysmyelination in mice with oligodendrocyte-specific deletion of Npc1 . (A) (Top panel) FluoroMyelin staining of the corpus callosum of Npc1 flox/− , CNP Cre/+ and control mice at P16. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (Bottom two panels) MBP immunofluorescence in forebrain sagittal sections of Npc1 flox/− , CNP Cre/+ mice and controls at P16 and 7 weeks. Bar, 500 µm. Area highlighted by white rectangle is enlarged in the inset in the upper right of each panel. (B) Western blots of myelin-specific proteins and neurofilament protein (NF-200) from brainstem and cerebral cortex homogenates of P16 Npc1 flox/− , CNP Cre/+ mice and controls. GAPDH controls for loading. (C) MBP and neurofilament protein (NF) co-staining in the corpus callosum of P16 Npc1 flox/− , CNP Cre/+ and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (D) Electron microscopy of the corpus callosum of a P16 Npc1 flox/− , CNP Cre/+ and control mice. Bar, 50 nm.

    Article Snippet: Antibodies used were rat anti-MBP (1∶2000, Abcam), mouse anti-CNP (1∶2000, Millipore), mouse anti-MAG (1∶5000, Millipore), mouse anti-Neurofilament 200 (1∶5000, Millipore), rabbit anti-NG2 (1∶1000, Millipore), rabbit anti-GAPDH (1∶5000, Santa Cruz), mouse anti-Cre (1∶1000, Millipore), rabbit anti-GFAP (1∶5000, Dako), mouse anti-PSA-NCAM (1∶1000, Millipore), goat anti-Jagged1(1∶1000, Santa Cruz) and rabbit anti-Lingo1 (1∶1000, Abcam).

    Techniques: Mouse Assay, Staining, Immunofluorescence, Western Blot, Electron Microscopy

    Atpif 1 deficiency causes a decline in the retinal expression of OPA1. a , b Fluorescent IHC of whole-mount 72 hpf zebrafish, stained with anti-OPA1 antibody. Representative images ( a ) and quantification of OPA1 fluorescence intensity ( b ) are reported, showing a reduction in the OPA1 expression levels in the brain and retina of pnt tq209 larvae (OPA1 fluorescence (A.U.), Sb: 1.00 ± 0.01, pnt tq209 : 0.70 ± 0.09; results are presented as mean ± S.E.M. ( n = 3)). c , d Analysis of eye morphology in Sb and pnt tq209 zebrafish obtained through anti-acetylated α-tubulin IHC of whole-mount 72 hpf zebrafish. No major morphological differences were observed between normal and mutant larvae, as shown in the prototypical images ( c ) and relative quantification of eye size ( d ) (eye size (pixels), Sb: 2.58 ± 0.10, pnt tq209 : 2.53 ± 0.06; results are presented as mean ± S.E.M. ( n = 10)). e , f Quantification of OPA1 levels via western blotting analysis in 72 hpf wild type and pnt tq209 larvae. The representative membrane blotted for both OPA1 isoform ( e ) and the bar chart ( f ) show a significant decrease in the levels of the short isoform of the protein in pnt tq209 larvae. g – j (OPA1 band density relative to ACTB, OPA1 long isoform Sb: 1.00 ± 0.01, pnt tq209 :0.97 ± 0.01; OPA1 short isoform Sb: 1.00 ± 0.01, pnt tq209 :0.84 ± 0.01; results are presented as mean ± S.E.M. ( n = 3), Quantitative western blot analysis of OPA1 levels in the brain (frontal cortex/hippocampus) ( j , k ) and isolated optic nerve ( l , m ) of WT and Atpif1 −/− mice. Representative blots ( j , l ) and quantitated band densities relative to GAPDH ( k , m ) are reported. Significantly lower levels of short and long OPA1 isoforms expression were found in the brain and, specifically, in the optic nerve of mutant mice (OPA1 band density relative to GAPDH, OPA1 long isoform WT: 1.00 ± 0.01, Atpif1 −/− : 0.52 ± 0.01 in the frontal cortex/hippocampus; in the optic nerve, WT: 1.00 ± 0.05, Atpif1 −/− : 0.59 ± 0.09; in the frontal cortex/hippocampus OPA1 short isoform WT: 1.00 ± 0.01, Atpif 1−/− : 0.68 ± 0.01; in the optic nerve, WT: 1.00 ± 0.05, Atpif1 −/− : 0.548 ± 0.01; results are presented as mean ± S.E.M. ( n = 3). FB forebrain, HB hindbrain, MB midbrain, NR neural retina, L lens

    Journal: Cell Death & Disease

    Article Title: Reduction of the ATPase inhibitory factor 1 (IF1) leads to visual impairment in vertebrates

    doi: 10.1038/s41419-018-0578-x

    Figure Lengend Snippet: Atpif 1 deficiency causes a decline in the retinal expression of OPA1. a , b Fluorescent IHC of whole-mount 72 hpf zebrafish, stained with anti-OPA1 antibody. Representative images ( a ) and quantification of OPA1 fluorescence intensity ( b ) are reported, showing a reduction in the OPA1 expression levels in the brain and retina of pnt tq209 larvae (OPA1 fluorescence (A.U.), Sb: 1.00 ± 0.01, pnt tq209 : 0.70 ± 0.09; results are presented as mean ± S.E.M. ( n = 3)). c , d Analysis of eye morphology in Sb and pnt tq209 zebrafish obtained through anti-acetylated α-tubulin IHC of whole-mount 72 hpf zebrafish. No major morphological differences were observed between normal and mutant larvae, as shown in the prototypical images ( c ) and relative quantification of eye size ( d ) (eye size (pixels), Sb: 2.58 ± 0.10, pnt tq209 : 2.53 ± 0.06; results are presented as mean ± S.E.M. ( n = 10)). e , f Quantification of OPA1 levels via western blotting analysis in 72 hpf wild type and pnt tq209 larvae. The representative membrane blotted for both OPA1 isoform ( e ) and the bar chart ( f ) show a significant decrease in the levels of the short isoform of the protein in pnt tq209 larvae. g – j (OPA1 band density relative to ACTB, OPA1 long isoform Sb: 1.00 ± 0.01, pnt tq209 :0.97 ± 0.01; OPA1 short isoform Sb: 1.00 ± 0.01, pnt tq209 :0.84 ± 0.01; results are presented as mean ± S.E.M. ( n = 3), Quantitative western blot analysis of OPA1 levels in the brain (frontal cortex/hippocampus) ( j , k ) and isolated optic nerve ( l , m ) of WT and Atpif1 −/− mice. Representative blots ( j , l ) and quantitated band densities relative to GAPDH ( k , m ) are reported. Significantly lower levels of short and long OPA1 isoforms expression were found in the brain and, specifically, in the optic nerve of mutant mice (OPA1 band density relative to GAPDH, OPA1 long isoform WT: 1.00 ± 0.01, Atpif1 −/− : 0.52 ± 0.01 in the frontal cortex/hippocampus; in the optic nerve, WT: 1.00 ± 0.05, Atpif1 −/− : 0.59 ± 0.09; in the frontal cortex/hippocampus OPA1 short isoform WT: 1.00 ± 0.01, Atpif 1−/− : 0.68 ± 0.01; in the optic nerve, WT: 1.00 ± 0.05, Atpif1 −/− : 0.548 ± 0.01; results are presented as mean ± S.E.M. ( n = 3). FB forebrain, HB hindbrain, MB midbrain, NR neural retina, L lens

    Article Snippet: The membrane was blocked in 3% non-fat dry milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween 20 (Sigma-Aldrich), pH 7.5) for 1 h and then incubated with the appropriate diluted primary antibody at 4 °C overnight: mouse anti-OPA1 (1:1000; BD Biosciences, 612607), mouse anti-GAPDH (1:10000; Abcam, ab 8245).

    Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence, Mutagenesis, Western Blot, Isolation, Mouse Assay