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  • 99
    Millipore monoclonal anti beta tubulin antibody
    Monoclonal Anti Beta Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5025 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore α tubulin
    Hh components accumulate in Dync2h1 mutant cilia Localization of Smo ( a, b ), Gli2 ( c, d ) and Ptch1 ( e, f ) to the primary cilium in wild-type and Dync2h1 lln/lln MEFs ( a, c, e ) and E10.5 neural tube ( b, d, f ). ( a ) Smo (green) was enriched in cilia of wild-type MEFs only after exposure to Shh. Smo was enriched in cilia of Dync2h1 lln/lln mutant cells even in the absence of Shh. ( b ) Smo was enriched in cilia of ventral neural progenitors in wild-type. Smo was strongly enriched in primary cilia of Dync2h1 lln/lln neural progenitors at all dorsal-ventral levels. ( c ) Gli2 (green) localized to the tips of cilia in wild-type MEFs and accumulated further after Shh treatment. Gli2 levels were elevated along the axoneme of Dync2h1 lln/lln mutant MEF cilia. ( d ) Gli2 was elevated in the cilia of Dync2h1 lln/lln neural progenitors. ( e ) Low amounts of endogenous Ptch1 (green) were detected near the base and along the length of primary cilia in wild-type MEFs only in the absence of Shh, whereas Ptch1 was strongly enriched along the axoneme of Dync2h1 lln/lln cilia in unstimulated cells; strong Ptch1 immunofluorescence remained near the base of the cilium after stimulation with Shh. ( f ) Ptch1 appeared localized to the cytoplasm of wild-type neural progenitors, and was strongly enriched in cilia throughout the neural tube in Dync2h1 lln/lln mutants. Acetylated <t>α-tubulin</t> (red) marks cilia in ( a, c, e ); ( b, d, f ) are ventral views of transverse sections through the ventral half of the neural tube at the level of the forelimb. Scale bars represent 500 nm ( a, c, e ), 25 μm ( b, d, f ) and 10 μm (insets b, d, f ).
    α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21797 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti α tubulin
    KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and <t>anti-tubulin</t> immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.
    Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11558 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tubulin
    KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and <t>anti-tubulin</t> immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.
    Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11549 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology α tubulin
    PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . <t>α-tubulin</t> was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.
    α Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 7178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti tubulin
    PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . <t>α-tubulin</t> was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.
    Anti Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc α tubulin
    PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . <t>α-tubulin</t> was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.
    α Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti α tubulin
    PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . <t>α-tubulin</t> was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.
    Mouse Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gamma tubulin antibody
    PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . <t>α-tubulin</t> was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.
    Anti Gamma Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam α tubulin
    PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . <t>α-tubulin</t> was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.
    α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β tubulin
    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). <t>β-Tubulin</t> (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).
    β Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam alpha tubulin
    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). <t>β-Tubulin</t> (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).
    Alpha Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti tubulin acetylated antibody
    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). <t>β-Tubulin</t> (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).
    Monoclonal Anti Tubulin Acetylated Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3808 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tubulin
    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). <t>β-Tubulin</t> (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).
    Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2637 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology tubulin
    C-terminal <t>tubulin</t> tail mutants increase microtubule dynamics and modulate cell mechanic-dependent activities. (a-d) HeLa cells were transfected with TUBA1A constructs and plated on 50kPa hydrogel. (a) Representative alignment of microtubule in cells. (b) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin in cells. (c) Proliferation rate of cells. (d) Cell velocity (left) and speed (right) of cells. In all the panels n > 50 cells from 3 independent experiments were analyzed. *P
    Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 3376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology β tubulin
    PGI 2 mimics iloprost-induced contractile apparatus and connexin 43 protein expression. ( A ) Effect of PGI 2 on PKA activity. Human uSMCs were treated for 20 min with PGI 2 or iloprost at the concentrations indicated and subjected to in vitro PKA assay. The phosphorylated kemptide gel is shown. ( B and C ) Effect of PGI 2 on contractile apparatus protein and connexin 43 expression. ( B ) Human uSMCs were treated with PGI 2 every 1.5 h up to 6 h or with a single treatment of iloprost for 6 h at the concentrations indicated. Western blots were performed with antibodies against SM2-MHC, calponin, <t>β-tubulin,</t> and GAPDH. ( C ) uSMCs were subjected to a single treatment of 0.6 μmol/l PGI 2 for the indicated times. Western blot analysis was performed with antibodies against SM2-MHC, h-caldesmon, connexin 43, and β-tubulin as a loading control.
    β Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 4138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti acetylated tubulin antibody
    Reduced centrosomal <t>γ-tubulin</t> staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.
    Monoclonal Anti Acetylated Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2829 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti α tubulin
    Reduced centrosomal <t>γ-tubulin</t> staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.
    Anti α Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Hh components accumulate in Dync2h1 mutant cilia Localization of Smo ( a, b ), Gli2 ( c, d ) and Ptch1 ( e, f ) to the primary cilium in wild-type and Dync2h1 lln/lln MEFs ( a, c, e ) and E10.5 neural tube ( b, d, f ). ( a ) Smo (green) was enriched in cilia of wild-type MEFs only after exposure to Shh. Smo was enriched in cilia of Dync2h1 lln/lln mutant cells even in the absence of Shh. ( b ) Smo was enriched in cilia of ventral neural progenitors in wild-type. Smo was strongly enriched in primary cilia of Dync2h1 lln/lln neural progenitors at all dorsal-ventral levels. ( c ) Gli2 (green) localized to the tips of cilia in wild-type MEFs and accumulated further after Shh treatment. Gli2 levels were elevated along the axoneme of Dync2h1 lln/lln mutant MEF cilia. ( d ) Gli2 was elevated in the cilia of Dync2h1 lln/lln neural progenitors. ( e ) Low amounts of endogenous Ptch1 (green) were detected near the base and along the length of primary cilia in wild-type MEFs only in the absence of Shh, whereas Ptch1 was strongly enriched along the axoneme of Dync2h1 lln/lln cilia in unstimulated cells; strong Ptch1 immunofluorescence remained near the base of the cilium after stimulation with Shh. ( f ) Ptch1 appeared localized to the cytoplasm of wild-type neural progenitors, and was strongly enriched in cilia throughout the neural tube in Dync2h1 lln/lln mutants. Acetylated α-tubulin (red) marks cilia in ( a, c, e ); ( b, d, f ) are ventral views of transverse sections through the ventral half of the neural tube at the level of the forelimb. Scale bars represent 500 nm ( a, c, e ), 25 μm ( b, d, f ) and 10 μm (insets b, d, f ).

    Journal: Nature genetics

    Article Title: Complex Interactions Between Genes Controlling Trafficking in Primary Cilia

    doi: 10.1038/ng.832

    Figure Lengend Snippet: Hh components accumulate in Dync2h1 mutant cilia Localization of Smo ( a, b ), Gli2 ( c, d ) and Ptch1 ( e, f ) to the primary cilium in wild-type and Dync2h1 lln/lln MEFs ( a, c, e ) and E10.5 neural tube ( b, d, f ). ( a ) Smo (green) was enriched in cilia of wild-type MEFs only after exposure to Shh. Smo was enriched in cilia of Dync2h1 lln/lln mutant cells even in the absence of Shh. ( b ) Smo was enriched in cilia of ventral neural progenitors in wild-type. Smo was strongly enriched in primary cilia of Dync2h1 lln/lln neural progenitors at all dorsal-ventral levels. ( c ) Gli2 (green) localized to the tips of cilia in wild-type MEFs and accumulated further after Shh treatment. Gli2 levels were elevated along the axoneme of Dync2h1 lln/lln mutant MEF cilia. ( d ) Gli2 was elevated in the cilia of Dync2h1 lln/lln neural progenitors. ( e ) Low amounts of endogenous Ptch1 (green) were detected near the base and along the length of primary cilia in wild-type MEFs only in the absence of Shh, whereas Ptch1 was strongly enriched along the axoneme of Dync2h1 lln/lln cilia in unstimulated cells; strong Ptch1 immunofluorescence remained near the base of the cilium after stimulation with Shh. ( f ) Ptch1 appeared localized to the cytoplasm of wild-type neural progenitors, and was strongly enriched in cilia throughout the neural tube in Dync2h1 lln/lln mutants. Acetylated α-tubulin (red) marks cilia in ( a, c, e ); ( b, d, f ) are ventral views of transverse sections through the ventral half of the neural tube at the level of the forelimb. Scale bars represent 500 nm ( a, c, e ), 25 μm ( b, d, f ) and 10 μm (insets b, d, f ).

    Article Snippet: In all experiments, ciliary microtubules were marked by expression of acetylated α-tubulin (mouse, 1:5000, Sigma Aldrich).

    Techniques: Mutagenesis, Immunofluorescence

    Ift172 is a dominant suppressor of Dync2h1 ( a ), E10.5 embryos and transverse sections through the caudal neural tube of wild-type, Dync2h1 lln/lln and Dync2h1 lln/lln Ift172 avc1 /+ embryos. Specification of floor plate (FoxA2, green), V3 progenitors (Nkx2.2, red) and motor neurons (HB9, green) were rescued in Dync2h1 lln/lln Ift172 avc1 /+ embryos. Scale bars represent 100 μm. ( b ), Dync2h1 lln/lln Ift172 avc1 /+ mutants survive to at least E16.5 (n=5). Scale bar is 5 mm. ( c ), Right forelimbs and digits of embryos in ( b ) stained with Alcian blue (cartilage) and Alizarin red (bone) staining shows incomplete penetrance of polydactyly in Dync2h1 lln/lln Ift172 avc1 /+ embryos at E16.5. ( d ), Scanning electron micrographs of cilia from the neural tube at E10.5 showing near-normal morphology of Dync2h1 lln/lln Ift172 avc1 /+ mutant cilia (quantitation in Supplementary Table 1 ). Scale bar is 500 nm. IFT88 (green, e ) and Smo (green, f ) and Gli2 (red, g ) localize normally in primary cilia of MEFs derived from Dync2h1 lln/lln Ift172 avc1 /+ embryos. Acetylated α-tubulin (red) marks cilia in ( e-g ). Scale bars represent 1 μm in ( e-g ).

    Journal: Nature genetics

    Article Title: Complex Interactions Between Genes Controlling Trafficking in Primary Cilia

    doi: 10.1038/ng.832

    Figure Lengend Snippet: Ift172 is a dominant suppressor of Dync2h1 ( a ), E10.5 embryos and transverse sections through the caudal neural tube of wild-type, Dync2h1 lln/lln and Dync2h1 lln/lln Ift172 avc1 /+ embryos. Specification of floor plate (FoxA2, green), V3 progenitors (Nkx2.2, red) and motor neurons (HB9, green) were rescued in Dync2h1 lln/lln Ift172 avc1 /+ embryos. Scale bars represent 100 μm. ( b ), Dync2h1 lln/lln Ift172 avc1 /+ mutants survive to at least E16.5 (n=5). Scale bar is 5 mm. ( c ), Right forelimbs and digits of embryos in ( b ) stained with Alcian blue (cartilage) and Alizarin red (bone) staining shows incomplete penetrance of polydactyly in Dync2h1 lln/lln Ift172 avc1 /+ embryos at E16.5. ( d ), Scanning electron micrographs of cilia from the neural tube at E10.5 showing near-normal morphology of Dync2h1 lln/lln Ift172 avc1 /+ mutant cilia (quantitation in Supplementary Table 1 ). Scale bar is 500 nm. IFT88 (green, e ) and Smo (green, f ) and Gli2 (red, g ) localize normally in primary cilia of MEFs derived from Dync2h1 lln/lln Ift172 avc1 /+ embryos. Acetylated α-tubulin (red) marks cilia in ( e-g ). Scale bars represent 1 μm in ( e-g ).

    Article Snippet: In all experiments, ciliary microtubules were marked by expression of acetylated α-tubulin (mouse, 1:5000, Sigma Aldrich).

    Techniques: Staining, Mutagenesis, Quantitation Assay, Derivative Assay

    Neural patterning and cilia morphology in Dync2h1 lln Ift122 sopb embryos ( a ), In contrast to the lack of ventral neural cell types in Dync2h1 lln/lln mutants, both Ift122 sopb/sopb single and Dync2h1 lln/lln Ift122 sopb/sopb double mutants specify floor plate (FoxA2, green), V3 progenitors (Nkx2.2, red) and motor neurons (HB9, green) in the lumbar neural tube. Scale bars represent 100 μm. ( b ), Scanning electron micrographs of neural tube cilia from the neural tube of E10.5 Ift122 sopb/sopb and Dync2h1 lln/lln Ift122 sopb/sopb embryos. The distal ends of Ift122 sopb/sopb mutant cilia appeared swollen. Dync2h1 lln/lln Ift122 sopb/sopb mutant cilia were similar in diameter to Ift122 sopb/sopb but were shorter than either Dync2h1 lln/lln or Ift122 sopb/sopb single mutants (See Supplementary Table 1 ). Scale bars represent 500 nm. ( c ), IFT88 (green) accumulates specifically at the distal tips of both Ift122 sopb/sopb and Dync2h1 lln/lln Ift122 sopb/sopb mutant MEF cilia. Acetylated α-tubulin staining (red) marks primary cilia. Localization of Smo ( d , green) and Gli2 ( e , green) in the cilia of Ift122 sopb/sopb and Dync2h1 lln/lln Ift122 sopb/sopb mutant MEFs. Acetylated α-tubulin (red) marks cilia. ( f ), Dync2h1 protein is present at the base of the cilium and along the ciliary axoneme in wild-type cells. In Ift122 sopb/sopb mutant cilia, Dync2h1 localization accumulates mainly at the base of the cilium. Orientation for ( b-f ) is distal tip up. Scale bars are 1 μm ( d-f ).

    Journal: Nature genetics

    Article Title: Complex Interactions Between Genes Controlling Trafficking in Primary Cilia

    doi: 10.1038/ng.832

    Figure Lengend Snippet: Neural patterning and cilia morphology in Dync2h1 lln Ift122 sopb embryos ( a ), In contrast to the lack of ventral neural cell types in Dync2h1 lln/lln mutants, both Ift122 sopb/sopb single and Dync2h1 lln/lln Ift122 sopb/sopb double mutants specify floor plate (FoxA2, green), V3 progenitors (Nkx2.2, red) and motor neurons (HB9, green) in the lumbar neural tube. Scale bars represent 100 μm. ( b ), Scanning electron micrographs of neural tube cilia from the neural tube of E10.5 Ift122 sopb/sopb and Dync2h1 lln/lln Ift122 sopb/sopb embryos. The distal ends of Ift122 sopb/sopb mutant cilia appeared swollen. Dync2h1 lln/lln Ift122 sopb/sopb mutant cilia were similar in diameter to Ift122 sopb/sopb but were shorter than either Dync2h1 lln/lln or Ift122 sopb/sopb single mutants (See Supplementary Table 1 ). Scale bars represent 500 nm. ( c ), IFT88 (green) accumulates specifically at the distal tips of both Ift122 sopb/sopb and Dync2h1 lln/lln Ift122 sopb/sopb mutant MEF cilia. Acetylated α-tubulin staining (red) marks primary cilia. Localization of Smo ( d , green) and Gli2 ( e , green) in the cilia of Ift122 sopb/sopb and Dync2h1 lln/lln Ift122 sopb/sopb mutant MEFs. Acetylated α-tubulin (red) marks cilia. ( f ), Dync2h1 protein is present at the base of the cilium and along the ciliary axoneme in wild-type cells. In Ift122 sopb/sopb mutant cilia, Dync2h1 localization accumulates mainly at the base of the cilium. Orientation for ( b-f ) is distal tip up. Scale bars are 1 μm ( d-f ).

    Article Snippet: In all experiments, ciliary microtubules were marked by expression of acetylated α-tubulin (mouse, 1:5000, Sigma Aldrich).

    Techniques: Mutagenesis, Staining

    Cilia morphology in Dync2h1 lln/lln Ift122 sopb /+ compound mutants ( a ), SEM analysis of neural tube primary cilia show the more normal length and width of Dync2h1 lln/lln Ift122 sopb /+ mutants compared to Dync2h1 lln/lln . Quantitation in Supplementary Table 1 . Scale bars are 500 nm. ( b-d ), Localization of IFT88 ( b , green), Smo ( c , green) and Gli2 ( d , green) in cilia (acetylated α-tubulin, red) appear normal in Dync2h1 lln/lln Ift122 sopb /+ mutants. Scale bars are 500 nm ( b-d ). ( e ) Model of the trafficking of mammalian IFT and Hh pathway proteins in the primary cilium, shown in the absence of Hh ligand. In wild-type cells, IFT directs the formation of cilia, which accumulate a basal level of Gli2 at cilia tips, while Smo traffics through the cilium at a low basal rate. Loss of retrograde motor in Dync2h1 lln/lln mutant cilia leads to the accumulation of IFT particles and blocks the movement of both Smo and Gli2 out of the cilium. In Ift122 sopb/sopb mutants, Dync2h1 protein fails to enter the cilium, leading to the accumulation of IFT-B particles. Loss of IFT122 also results in the accumulation of Gli2 but does not affect Smo trafficking. Decreased anterograde ciliary trafficking in Dync2h1 lln/lln Ift122 sopb /+ suppresses the Dync2h1 lln/lln phenotype and permits normal transport of both Smo and Gli2 through the cilium.

    Journal: Nature genetics

    Article Title: Complex Interactions Between Genes Controlling Trafficking in Primary Cilia

    doi: 10.1038/ng.832

    Figure Lengend Snippet: Cilia morphology in Dync2h1 lln/lln Ift122 sopb /+ compound mutants ( a ), SEM analysis of neural tube primary cilia show the more normal length and width of Dync2h1 lln/lln Ift122 sopb /+ mutants compared to Dync2h1 lln/lln . Quantitation in Supplementary Table 1 . Scale bars are 500 nm. ( b-d ), Localization of IFT88 ( b , green), Smo ( c , green) and Gli2 ( d , green) in cilia (acetylated α-tubulin, red) appear normal in Dync2h1 lln/lln Ift122 sopb /+ mutants. Scale bars are 500 nm ( b-d ). ( e ) Model of the trafficking of mammalian IFT and Hh pathway proteins in the primary cilium, shown in the absence of Hh ligand. In wild-type cells, IFT directs the formation of cilia, which accumulate a basal level of Gli2 at cilia tips, while Smo traffics through the cilium at a low basal rate. Loss of retrograde motor in Dync2h1 lln/lln mutant cilia leads to the accumulation of IFT particles and blocks the movement of both Smo and Gli2 out of the cilium. In Ift122 sopb/sopb mutants, Dync2h1 protein fails to enter the cilium, leading to the accumulation of IFT-B particles. Loss of IFT122 also results in the accumulation of Gli2 but does not affect Smo trafficking. Decreased anterograde ciliary trafficking in Dync2h1 lln/lln Ift122 sopb /+ suppresses the Dync2h1 lln/lln phenotype and permits normal transport of both Smo and Gli2 through the cilium.

    Article Snippet: In all experiments, ciliary microtubules were marked by expression of acetylated α-tubulin (mouse, 1:5000, Sigma Aldrich).

    Techniques: Quantitation Assay, Mutagenesis

    Mutations in Dync2h1 disrupt Shh-dependent neural patterning and cilia morphology ( a ), Mutations in Dync2h1 lead to the absence of Shh-dependent cell types in the E10.5 neural tube. In Dync2h1 lln/lln mutants, floor plate (FoxA2, green) and V3 progenitor (Nkx2.2, red) domains are not specified, and motor neurons (HB9, green) are present only in the caudal neural tube (shown here); dorsal up. Scale bars represent 100 μm. ( b ), Scanning electron micrographs show that neural tube primary cilia in Dync2h1 lln/lln mutants are bloated; dimensions are given in Supplementary Table1 . Scale bars represent 500 nm. ( c ), IFT88 (green) in cilia of serum-starved wild-type MEFs is enriched at the base and the tip of the cilium, marked with acetylated α-tubulin (red). In Dync2h1 lln/lln mutant MEFs, the amount of IFT88 in the cilium is increased and is found all along the axoneme. Quantitation is in Supplementary Table 2 . Scale bars represent 1 μm ( c ).

    Journal: Nature genetics

    Article Title: Complex Interactions Between Genes Controlling Trafficking in Primary Cilia

    doi: 10.1038/ng.832

    Figure Lengend Snippet: Mutations in Dync2h1 disrupt Shh-dependent neural patterning and cilia morphology ( a ), Mutations in Dync2h1 lead to the absence of Shh-dependent cell types in the E10.5 neural tube. In Dync2h1 lln/lln mutants, floor plate (FoxA2, green) and V3 progenitor (Nkx2.2, red) domains are not specified, and motor neurons (HB9, green) are present only in the caudal neural tube (shown here); dorsal up. Scale bars represent 100 μm. ( b ), Scanning electron micrographs show that neural tube primary cilia in Dync2h1 lln/lln mutants are bloated; dimensions are given in Supplementary Table1 . Scale bars represent 500 nm. ( c ), IFT88 (green) in cilia of serum-starved wild-type MEFs is enriched at the base and the tip of the cilium, marked with acetylated α-tubulin (red). In Dync2h1 lln/lln mutant MEFs, the amount of IFT88 in the cilium is increased and is found all along the axoneme. Quantitation is in Supplementary Table 2 . Scale bars represent 1 μm ( c ).

    Article Snippet: In all experiments, ciliary microtubules were marked by expression of acetylated α-tubulin (mouse, 1:5000, Sigma Aldrich).

    Techniques: Mutagenesis, Quantitation Assay

    KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.

    Journal: PLoS Pathogens

    Article Title: The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein

    doi: 10.1371/journal.ppat.1006937

    Figure Lengend Snippet: KSHV ORF20 encodes three isoforms. (A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.

    Article Snippet: Rabbit anti-c-myc (# C3956), mouse anti-tubulin (# T6199-200UL), and mouse anti-beta-actin (#A5441) antibodies were purchased from Sigma-Aldrich.

    Techniques: Plasmid Preparation, Construct, Expressing, Transfection, Polyacrylamide Gel Electrophoresis

    The interaction of ORF20 with OASL is conserved among the members of the UL24 family. (A) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, coverslips were fixed in PFA and processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (B) 293T cells were transfected with myc-tagged UL24, UL76, M76, or ORF20WT. Lysates were prepared 24 h later, separated by SDS-PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Data are representative of four independent experiments. (C) 293T cells were transfected with the indicated myc-tagged KSHV ORF20 form or MHV68 ORF20. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with an anti-myc antibody. Data are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged UL24 homologs MCMV M76, HCMV UL76, or HSV-1 UL24, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein

    doi: 10.1371/journal.ppat.1006937

    Figure Lengend Snippet: The interaction of ORF20 with OASL is conserved among the members of the UL24 family. (A) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, coverslips were fixed in PFA and processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (B) 293T cells were transfected with myc-tagged UL24, UL76, M76, or ORF20WT. Lysates were prepared 24 h later, separated by SDS-PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Data are representative of four independent experiments. (C) 293T cells were transfected with the indicated myc-tagged KSHV ORF20 form or MHV68 ORF20. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with an anti-myc antibody. Data are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged UL24 homologs MCMV M76, HCMV UL76, or HSV-1 UL24, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of two independent experiments.

    Article Snippet: Rabbit anti-c-myc (# C3956), mouse anti-tubulin (# T6199-200UL), and mouse anti-beta-actin (#A5441) antibodies were purchased from Sigma-Aldrich.

    Techniques: Transfection, Plasmid Preparation, Immunofluorescence, SDS Page, Immunoprecipitation

    PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . α-tubulin was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: PINK1 protein levels after ionomycin or Bay K 8644 treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with the vehicle (0.05% (v/v) ethanol), with ionomycin or Bay K 8644 for 24 h, lysates prepared and Western-blotting performed. Blots were probed with antibodies against PINK1 . α-tubulin was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity. Molecular mass is indicated in kD next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Western Blot

    Increase of PINK1 gene expression, and not its stabilization with chaperones, after the CCCP exposure. (A) SH-SY5Y cells were exposed with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol) at different times, and PINK1 mRNA levels measured by reverse transcription and quantitative PCR. Relative expression was determined using GAPDH as housekeeping gene (#p > 0.05; **p ≤ 0.01; ***p ≤ 0.001). (B and C) SH-SY5Y cells were preincubated 1 h with 5 μg/ml Act. D, 100 μg/ml CHX or vehicle (0.1% (v/v) DMSO), exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, harvested by trypsinization and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (B) Representative blot of at least three independent experiments. (C) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). (D–F) SH-SY5Y cells were preincubated 1 h with 5 μM MG-132 or 100 nM Baf. A1, exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, incubated with 1 μM 17-AAG 3 h before collecting cells, harvested by trypsinization and lysed. The protein levels of PINK1 and COX IV were determined by Western-blotting. α-tubulin expression was used as a loading control. (D) Representative blot of at least three independent experiments. (E) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). (F) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: Increase of PINK1 gene expression, and not its stabilization with chaperones, after the CCCP exposure. (A) SH-SY5Y cells were exposed with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol) at different times, and PINK1 mRNA levels measured by reverse transcription and quantitative PCR. Relative expression was determined using GAPDH as housekeeping gene (#p > 0.05; **p ≤ 0.01; ***p ≤ 0.001). (B and C) SH-SY5Y cells were preincubated 1 h with 5 μg/ml Act. D, 100 μg/ml CHX or vehicle (0.1% (v/v) DMSO), exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, harvested by trypsinization and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (B) Representative blot of at least three independent experiments. (C) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). (D–F) SH-SY5Y cells were preincubated 1 h with 5 μM MG-132 or 100 nM Baf. A1, exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, incubated with 1 μM 17-AAG 3 h before collecting cells, harvested by trypsinization and lysed. The protein levels of PINK1 and COX IV were determined by Western-blotting. α-tubulin expression was used as a loading control. (D) Representative blot of at least three independent experiments. (E) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). (F) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay, Western Blot, Incubation

    c-Fos-independent expression of PINK1 after CCCP exposure. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 3 h with 10 μM CCCP, harvested by trypsinization and lysed. The protein levels of p-c-Fos (Ser32) were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). (C–F) SH-SY5Y cells were transfected with c-fos siRNA, PINK1 siRNA or scrambled control siRNA for 2 days and treated with 10 μM CCCP, harvested by trypsinization at different times and lysed. The protein levels of p-c-Fos (Ser32), c-Fos and PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments, from 3 hour-CCCP-treated cells. (D) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). (E) Representative blot of at least three independent experiments, from 24 hour-CCCP-treated cells. (F) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: c-Fos-independent expression of PINK1 after CCCP exposure. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 3 h with 10 μM CCCP, harvested by trypsinization and lysed. The protein levels of p-c-Fos (Ser32) were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). (C–F) SH-SY5Y cells were transfected with c-fos siRNA, PINK1 siRNA or scrambled control siRNA for 2 days and treated with 10 μM CCCP, harvested by trypsinization at different times and lysed. The protein levels of p-c-Fos (Ser32), c-Fos and PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments, from 3 hour-CCCP-treated cells. (D) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). (E) Representative blot of at least three independent experiments, from 24 hour-CCCP-treated cells. (F) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Expressing, Western Blot, Transfection

    LC3 levels after UPS/autophagy blockade or 17-AAG treatment. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM MG-132 or 100 nM Baf. A1, exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, incubated with 1 μM 17-AAG 3 h before collecting cells, harvested by trypsinization and lysed. The LC3-II/LC3-I ratio was determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: LC3 levels after UPS/autophagy blockade or 17-AAG treatment. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM MG-132 or 100 nM Baf. A1, exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, incubated with 1 μM 17-AAG 3 h before collecting cells, harvested by trypsinization and lysed. The LC3-II/LC3-I ratio was determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Incubation, Western Blot, Expressing

    Involvement of extracellular calcium in PINK1 levels. (A–D) Time courses of Ratio (F340/F380) in Fura-2 AM loaded SH-SY5Y cells to determine cytosolic calcium changes. (A) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells after the addition of 10 μM CCCP in Ca 2 + -free Locke's K25 buffer. (B) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells induced by 10 μM CCCP in complete Locke's K25 buffer (***p ≤ 0.001 between + Ca 2 + -CCCP-treated (n = 22) and − Ca 2 + -CCCP-treated cells (n = 12)). (C) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells after the treatment of 10 μM nifedipine and 10 μM CCCP in complete Locke's K25 buffer (*p ≤ 0.05 between + Ca 2 + -nifedipine-CCCP-treated (n = 10) and + Ca 2 + -CCCP-treated cells). (D) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells in the presence of 2 μM ω-CTX and 10 μM CCCP in complete Locke's K25 buffer (**p ≤ 0.01 between + Ca 2 + -ω-CTX-CCCP-treated (n = 31) and + Ca 2 + -CCCP-treated cells). (E and F) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM, exposed with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), harvested by trypsinization at different times and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (E) Representative blot of at least three independent experiments. (F) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; *p ≤ 0.05; ***p ≤ 0.001). (G and H) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM, exposed 6 or 24 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), mitochondrial isolated and Western-blotting performed. Tom20 was used as a mitochondrial loading control. (G) Representative blot of at least three independent experiments. (H) Densitometry of each band expressed in arbitrary units of intensity (**p ≤ 0.01; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: Involvement of extracellular calcium in PINK1 levels. (A–D) Time courses of Ratio (F340/F380) in Fura-2 AM loaded SH-SY5Y cells to determine cytosolic calcium changes. (A) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells after the addition of 10 μM CCCP in Ca 2 + -free Locke's K25 buffer. (B) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells induced by 10 μM CCCP in complete Locke's K25 buffer (***p ≤ 0.001 between + Ca 2 + -CCCP-treated (n = 22) and − Ca 2 + -CCCP-treated cells (n = 12)). (C) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells after the treatment of 10 μM nifedipine and 10 μM CCCP in complete Locke's K25 buffer (*p ≤ 0.05 between + Ca 2 + -nifedipine-CCCP-treated (n = 10) and + Ca 2 + -CCCP-treated cells). (D) Time course of [Ca 2 + ] cyt changes in SH-SY5Y cells in the presence of 2 μM ω-CTX and 10 μM CCCP in complete Locke's K25 buffer (**p ≤ 0.01 between + Ca 2 + -ω-CTX-CCCP-treated (n = 31) and + Ca 2 + -CCCP-treated cells). (E and F) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM, exposed with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), harvested by trypsinization at different times and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (E) Representative blot of at least three independent experiments. (F) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; *p ≤ 0.05; ***p ≤ 0.001). (G and H) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM, exposed 6 or 24 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), mitochondrial isolated and Western-blotting performed. Tom20 was used as a mitochondrial loading control. (G) Representative blot of at least three independent experiments. (H) Densitometry of each band expressed in arbitrary units of intensity (**p ≤ 0.01; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Western Blot, Expressing, Isolation

    CCCP-induced mitophagy is parallel to mitochondrial PINK1 localization. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), harvested by trypsinization at different times, mitochondria isolated and Western-blotting performed. Blots were probed with antibodies against PINK1 and LC3B. α-tubulin and Tom20 were used as a cytosolic and mitochondrial loading control, respectively. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. (C–F) SH-SY5Y cells were transfected with mCherry-Parkin or GFP-LC3 and exposed 6, 12 or 24 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), fixed and immunostained for Tom20 (green/red). (C and D) Representative immunofluorescence microphotographs of 6 hour treated-cells. The boxes highlight mitochondrial localization of mCherry-Parkin and GFP-LC3, respectively. (E) Percentages of cells with mCherry-Parkin on mitochondria, labeled with anti-Tom20 antibody (***p ≤ 0.001). (F) Percentages of cells with GFP-LC3 on mitochondria, labeled with anti-Tom20 antibody (***p ≤ 0.001). Scale bar represents 10 μm.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: CCCP-induced mitophagy is parallel to mitochondrial PINK1 localization. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), harvested by trypsinization at different times, mitochondria isolated and Western-blotting performed. Blots were probed with antibodies against PINK1 and LC3B. α-tubulin and Tom20 were used as a cytosolic and mitochondrial loading control, respectively. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. (C–F) SH-SY5Y cells were transfected with mCherry-Parkin or GFP-LC3 and exposed 6, 12 or 24 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), fixed and immunostained for Tom20 (green/red). (C and D) Representative immunofluorescence microphotographs of 6 hour treated-cells. The boxes highlight mitochondrial localization of mCherry-Parkin and GFP-LC3, respectively. (E) Percentages of cells with mCherry-Parkin on mitochondria, labeled with anti-Tom20 antibody (***p ≤ 0.001). (F) Percentages of cells with GFP-LC3 on mitochondria, labeled with anti-Tom20 antibody (***p ≤ 0.001). Scale bar represents 10 μm.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Isolation, Western Blot, Transfection, Immunofluorescence, Labeling

    Mitochondrial damage and autophagy induction in SH-SY5Y CCCP-treated cells. (A) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), lysed and CS activity measured. Data are expressed as nmol/min/mg protein (#p > 0.05; ***p ≤ 0.001). (B and C) SH-SY5Y cells were treated with 10 μM CCCP for 0, 3 or 24 h and lysates separated by SDS-PAGE and Western-blotting performed. Blots were probed with antibodies against two inner mitochondrial membrane proteins, COX IV and prohibitin 1. β-actin was used as a loading control. (B) Representative blot of at least three independent experiments. (C) Densitometry of each band expressed as % of control (#p > 0.05; *p ≤ 0.05; **p ≤ 0.01). (D and E) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, and stained with TMRM to assess Δψm by immunofluorescence. (D) Representative microphotographs of TMRM stain. Scale bar represents 10 μm. The arrows highlight cells with low Δψm. (E) TMRM fluorescence intensity per cell (in AU) by immunofluorescence (# p > 0.05; *** p ≤ 0.001). (F and G) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at different times and lysed. The ratio LC3-II/LC3-I was determined by Western-blotting. α-tubulin expression was used as a loading control. (F) Representative blot of at least three independent experiments. (G) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). Molecular mass is indicated in kilodaltons (kDa) next to the blots. Data were expressed as mean ± SEM; n = 3. (H and I) SH-SY5Y cells were transfected with mCherry-GFP-LC3B plasmid for 24 h and treated with 10 μM CCCP for 0, 3 or 24 h and fixed. (H) Representative immunofluorescence microphotographs. Autophagolysosomes and autophagosomes were labeled by red (mCherry-LC3B) and yellow puncta (mCherry-GFP-LC3B), respectively. The boxes highlight the pattern of each condition. (I) Percentages of mCherry (+) puncta per cell (#p > 0.05; ***p ≤ 0.001). Scale bar represents 10 μm.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: Mitochondrial damage and autophagy induction in SH-SY5Y CCCP-treated cells. (A) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), lysed and CS activity measured. Data are expressed as nmol/min/mg protein (#p > 0.05; ***p ≤ 0.001). (B and C) SH-SY5Y cells were treated with 10 μM CCCP for 0, 3 or 24 h and lysates separated by SDS-PAGE and Western-blotting performed. Blots were probed with antibodies against two inner mitochondrial membrane proteins, COX IV and prohibitin 1. β-actin was used as a loading control. (B) Representative blot of at least three independent experiments. (C) Densitometry of each band expressed as % of control (#p > 0.05; *p ≤ 0.05; **p ≤ 0.01). (D and E) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control) for 24 h, and stained with TMRM to assess Δψm by immunofluorescence. (D) Representative microphotographs of TMRM stain. Scale bar represents 10 μm. The arrows highlight cells with low Δψm. (E) TMRM fluorescence intensity per cell (in AU) by immunofluorescence (# p > 0.05; *** p ≤ 0.001). (F and G) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at different times and lysed. The ratio LC3-II/LC3-I was determined by Western-blotting. α-tubulin expression was used as a loading control. (F) Representative blot of at least three independent experiments. (G) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). Molecular mass is indicated in kilodaltons (kDa) next to the blots. Data were expressed as mean ± SEM; n = 3. (H and I) SH-SY5Y cells were transfected with mCherry-GFP-LC3B plasmid for 24 h and treated with 10 μM CCCP for 0, 3 or 24 h and fixed. (H) Representative immunofluorescence microphotographs. Autophagolysosomes and autophagosomes were labeled by red (mCherry-LC3B) and yellow puncta (mCherry-GFP-LC3B), respectively. The boxes highlight the pattern of each condition. (I) Percentages of mCherry (+) puncta per cell (#p > 0.05; ***p ≤ 0.001). Scale bar represents 10 μm.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Activity Assay, SDS Page, Western Blot, Staining, Immunofluorescence, Fluorescence, Expressing, Transfection, Plasmid Preparation, Labeling

    CCCP effect on PINK1 protein levels over time. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at different times and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). (C and D) SH-SY5Y cells were transfected with PINK1 siRNA or scrambled control siRNA for 3 days and treated with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at 24 h of treatment. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments. (D) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: CCCP effect on PINK1 protein levels over time. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at different times and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). (C and D) SH-SY5Y cells were transfected with PINK1 siRNA or scrambled control siRNA for 3 days and treated with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at 24 h of treatment. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments. (D) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Western Blot, Expressing, Transfection

    Mitophagy decreases after calcium chelation, but is a c-Fos-independent mechanism. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 24 h with 10 μM CCCP, harvested by trypsinization and lysed. The ratio LC3-II/LC3-I and protein levels of COX IV were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; **p ≤ 0.01). (C and D) SH-SY5Y cells were transfected with c-fos siRNA, PINK1 siRNA or scrambled control siRNA for 2 days and treated 24 h with 10 μM CCCP, harvested by trypsinization and lysed. The ratio LC3-II/LC3-I and protein levels of COX IV were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments. (D) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: Mitophagy decreases after calcium chelation, but is a c-Fos-independent mechanism. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 24 h with 10 μM CCCP, harvested by trypsinization and lysed. The ratio LC3-II/LC3-I and protein levels of COX IV were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; **p ≤ 0.01). (C and D) SH-SY5Y cells were transfected with c-fos siRNA, PINK1 siRNA or scrambled control siRNA for 2 days and treated 24 h with 10 μM CCCP, harvested by trypsinization and lysed. The ratio LC3-II/LC3-I and protein levels of COX IV were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments. (D) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Western Blot, Expressing, Transfection

    Nuclear recruitment of c-Fos after the CCCP treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at different times and lysed. The protein levels of p-c-Fos (Ser32) and c-Fos were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; *p ≤ 0.05; **p ≤ 0.01). (C–F) SH-SY5Y cells were exposed 3 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), fixed and immunostained for p-c-Fos (Ser32) or c-Fos (green) and Ho (blue). (C) Representative immunofluorescence microphotographs. The arrows highlight high nuclear intensity of p-c-Fos (Ser32). (D) Representative immunofluorescence microphotographs. The arrows highlight c-Fos nuclear staining. (E) Nuclear fluorescence intensity per cell (in AU), staining with anti-p-c-Fos (Ser32) antibody (***p ≤ 0.001). (F) Percentages of cells with nuclear c-Fos (***p ≤ 0.001). Scale bar represents 10 μm. Data were expressed as mean ± SEM; n = 200. (G and H) SH-SY5Y cells were exposed 3 h with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), nuclear isolated and Western-blotting performed. Lamin A/C was used as a nuclear loading control. (G) Representative blot of at least three independent experiments. (H) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: Nuclear recruitment of c-Fos after the CCCP treatment. (A and B) SH-SY5Y cells were exposed with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), harvested by trypsinization at different times and lysed. The protein levels of p-c-Fos (Ser32) and c-Fos were determined by Western-blotting. α-tubulin expression was used as a loading control. (A) Representative blot of at least three independent experiments. (B) Densitometry of each band expressed in arbitrary units of intensity (#p > 0.05; *p ≤ 0.05; **p ≤ 0.01). (C–F) SH-SY5Y cells were exposed 3 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), fixed and immunostained for p-c-Fos (Ser32) or c-Fos (green) and Ho (blue). (C) Representative immunofluorescence microphotographs. The arrows highlight high nuclear intensity of p-c-Fos (Ser32). (D) Representative immunofluorescence microphotographs. The arrows highlight c-Fos nuclear staining. (E) Nuclear fluorescence intensity per cell (in AU), staining with anti-p-c-Fos (Ser32) antibody (***p ≤ 0.001). (F) Percentages of cells with nuclear c-Fos (***p ≤ 0.001). Scale bar represents 10 μm. Data were expressed as mean ± SEM; n = 200. (G and H) SH-SY5Y cells were exposed 3 h with 10 μM CCCP, with vehicle (0.05% (v/v) ethanol) or without any treatment (control), nuclear isolated and Western-blotting performed. Lamin A/C was used as a nuclear loading control. (G) Representative blot of at least three independent experiments. (H) Densitometry of each band expressed in arbitrary units of intensity (***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Isolation

    PINK1 calcium-dependent gene expression. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 6 or 24 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), and PINK1 mRNA levels measured by reverse transcription and quantitative PCR. Relative expression was determined using GAPDH as housekeeping gene antibody. (A) Relative PINK1 mRNA expression after 6 hour CCCP exposure and calcium chelation (***p ≤ 0.001). (B) Relative PINK1 mRNA expression after 24 hours of CCCP treatment and calcium chelation (#p > 0.05). (C and D) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 24 h with 10 μM CCCP, harvested by trypsinization and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments. (D) Densitometry of each band expressed in arbitrary units of intensity (*p ≤ 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Journal: Neurobiology of Disease

    Article Title: Mitochondrial impairment increases FL-PINK1 levels by calcium-dependent gene expression

    doi: 10.1016/j.nbd.2013.10.021

    Figure Lengend Snippet: PINK1 calcium-dependent gene expression. (A and B) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 6 or 24 h with 10 μM CCCP or with vehicle (0.05% (v/v) ethanol), and PINK1 mRNA levels measured by reverse transcription and quantitative PCR. Relative expression was determined using GAPDH as housekeeping gene antibody. (A) Relative PINK1 mRNA expression after 6 hour CCCP exposure and calcium chelation (***p ≤ 0.001). (B) Relative PINK1 mRNA expression after 24 hours of CCCP treatment and calcium chelation (#p > 0.05). (C and D) SH-SY5Y cells were preincubated 1 h with 5 μM BAPTA-AM or 500 μM EGTA, exposed 24 h with 10 μM CCCP, harvested by trypsinization and lysed. The protein levels of PINK1 were determined by Western-blotting. α-tubulin expression was used as a loading control. (C) Representative blot of at least three independent experiments. (D) Densitometry of each band expressed in arbitrary units of intensity (*p ≤ 0.05; ***p ≤ 0.001). Molecular mass is indicated in kDa next to the blots. Data were expressed as mean ± SEM; n = 3.

    Article Snippet: Blots were probed with antibodies against PINK1 (clone BC100-494, Novus Biologicals, Southpark Way, Littleton, CO), subunit IV of cytochrome c oxidase (COX IV, ab14744, abcam), prohibitin 1 (#2426, Cell Signaling Technology, Beverly, MA), LC3B (#2775, Cell Signaling Technology, Beverly, MA), p-c-Fos (Ser32) (#5348, Cell Signaling Technology, Beverly, MA), c-Fos (#2250, Cell Signaling Technology, Beverly, MA), β-actin (ab8227, Abcam, Cambridge, UK), α-tubulin (clone TU-02, Santa Cruz Biotechnology, Santa Cruz, CA), Tom20 (clone F-10, Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (612162, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    SFN metabolites lowered the interactions among microtubule associated proteins leading to microtubule disruption and reduced resistance to PTX. a Immunofluorescence staining of Tau, α-tubulin and βIII-tubulin, XIAP showed the raising co-localization in cells and the changes of microtubule morphology treated with either 30 μM SFN-Cys or 30 μM SFN-NAC. Blue: DAPI-stained DNA; White arrows: normal microtubules; red arrows: the abnormal microtubules. Scale bars, 25 μm. The images in last row exhibited the zoom-in merged results. b Cells were treated with 30 μM SFN-Cys for 24 h. The binding of Tau to βIII-tubulin and binding of XIAP to α-tubulin was detected in A549/Taxol-R cells by forward and reverse co-immunoprecipitation (Co-IP). β-actin was used to be the loading controls for input proteins. c The dynamics of microtubules was measured by microtubule polymerization assay in vivo, and β-actin acted as the loading control. The expression of α-tubulin and β-tubulin was detected by Western blot with the treatment of 30 μM SFN-Cys for 24 h in A549/Taxol-R cells in soluble and insoluble cell lysate. The histogram showed the quantification of soluble and insoluble α-tubulin and β-tubulin. These results were from three independent experiments. d The expression of α-tubulin and β-tubulin was detected by Western blot with the treatment of 30 μM SFN-NAC for 24 h in A549/Taxol-R cells in soluble and insoluble cell lysate. The histogram showed the quantification of soluble and insoluble α-tubulin and β-tubulin. These results were from three independent experiments. e After treated with either 30 μM SFN-Cys or 30 μM SFN-NAC 24 h in A549/Taxol-R cells, then the cells were harvested and fixed, the cells pellets were cut into thin slices and microtubule structures were observed with a transmission electron microscope. f The expression of βIII-tubulin and α-tubulin was detected by Western blot after knockdown of βIII-tubulin and α-tubulin via siRNA in A549/Taxol-R cells. g Knockdown of βIII-tubulin and α-tubulin via RNA interference in A549/Taxol-R cells with/without 20 nM for 24 h, then the cells were harvested and the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. Data were shown as means ± SD from three separate experiments. * P

    Journal: Cell Death & Disease

    Article Title: Sulforaphane metabolites reduce resistance to paclitaxel via microtubule disruption

    doi: 10.1038/s41419-018-1174-9

    Figure Lengend Snippet: SFN metabolites lowered the interactions among microtubule associated proteins leading to microtubule disruption and reduced resistance to PTX. a Immunofluorescence staining of Tau, α-tubulin and βIII-tubulin, XIAP showed the raising co-localization in cells and the changes of microtubule morphology treated with either 30 μM SFN-Cys or 30 μM SFN-NAC. Blue: DAPI-stained DNA; White arrows: normal microtubules; red arrows: the abnormal microtubules. Scale bars, 25 μm. The images in last row exhibited the zoom-in merged results. b Cells were treated with 30 μM SFN-Cys for 24 h. The binding of Tau to βIII-tubulin and binding of XIAP to α-tubulin was detected in A549/Taxol-R cells by forward and reverse co-immunoprecipitation (Co-IP). β-actin was used to be the loading controls for input proteins. c The dynamics of microtubules was measured by microtubule polymerization assay in vivo, and β-actin acted as the loading control. The expression of α-tubulin and β-tubulin was detected by Western blot with the treatment of 30 μM SFN-Cys for 24 h in A549/Taxol-R cells in soluble and insoluble cell lysate. The histogram showed the quantification of soluble and insoluble α-tubulin and β-tubulin. These results were from three independent experiments. d The expression of α-tubulin and β-tubulin was detected by Western blot with the treatment of 30 μM SFN-NAC for 24 h in A549/Taxol-R cells in soluble and insoluble cell lysate. The histogram showed the quantification of soluble and insoluble α-tubulin and β-tubulin. These results were from three independent experiments. e After treated with either 30 μM SFN-Cys or 30 μM SFN-NAC 24 h in A549/Taxol-R cells, then the cells were harvested and fixed, the cells pellets were cut into thin slices and microtubule structures were observed with a transmission electron microscope. f The expression of βIII-tubulin and α-tubulin was detected by Western blot after knockdown of βIII-tubulin and α-tubulin via siRNA in A549/Taxol-R cells. g Knockdown of βIII-tubulin and α-tubulin via RNA interference in A549/Taxol-R cells with/without 20 nM for 24 h, then the cells were harvested and the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. Data were shown as means ± SD from three separate experiments. * P

    Article Snippet: Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA).

    Techniques: Immunofluorescence, Staining, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Polymerization Assay, In Vivo, Expressing, Western Blot, Transmission Assay, Microscopy, Flow Cytometry, Cytometry

    Combination of PTX with SFN metabolites showed a synergistic inhibition in A549/Taxol-R cells. a A549/Taxol-R cells were treated with (0, 5, 10, 15, 20, 25, 30, 35, 40 nM) combined with either 10 μM SFN-Cys or 10 μM SFN-NAC, respectively at the indicated concentrations for 24 h. Then, cell viability was determined by Cell Proliferation Assay Kit. b A549/Taxol-R cells were treated with PTX (20 nM), SFN-Cys (20 μM) or SFN-NAC (20 μM), SFN-Cys (10 μM) or SFN-NAC (10 μM) combined with PTX (10 nM), respectively for 24 h, then the cells were harvested and the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit (P: PTX 20 nM; C: SFN-Cys 20 μM; N: SFN-NAC 20 μM; PC: PTX 10 nM + SFN-Cys 10 μM; PN: PTX 10 nM + SFN-NAC 10 μM). c The histogram demonstrated the number of apoptotic cells in each group was detected by flow cytometry. d A549/Taxol-R cells was treated with both PTX (20 nM), SFN-Cys (20 μM) or SFN-NAC (20 μM), SFN-Cys (10 μM) or SFN-NAC (10 μM) combined with PTX (10 nM), respectively for 24 h, then recorded by Leica DMIRB microscope at ×40 magnification. e A549/Taxol-R cells were treated with PTX (20 nM), SFN-Cys (20 μM) or SFN-NAC (20 μM), SFN-Cys (10 μM) or SFN-NAC (10 μM) combined with PTX (10 nM) respectively for 24 h, then we harvested cells and viewed subcellular structures with a transmission electron microscope. Black arrows indicated sporadic vacuoles, white arrows indicated nucleic condensation. f The expression of Caspase-7, pro-Caspase-3 and cleaved-Caspase-3 was detected by Western blot in the groups (P: PTX 20 nM; C: SFN-Cys 20 μM; N: SFN-NAC 20 μM; PC: PTX 10 nM + SFN-Cys 10 μM; PN: PTX 10 nM + SFN-NAC 10 μM). g Cell viability was determined by Cell Proliferation Assay Kit. h Cells were harvested and the percentages of cell apoptosis were analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. i The histogram showed the number of apoptotic cells in each group. j Immunofluorescence staining of α-tubulin showed the changes of microtubule morphology treated with PTX and SFN-NAC in different groups. We also treated A549/Taxol-R cells with PTX and SFN-Cys in different groups, the results are the same as the combination of PTX and SFN-NAC (data not shown). Red: α-tubulin, Blue: DAPI-stained DNA; White arrows: normal microtubules; red arrows: the abnormal microtubules. Scale bars, 25 μm. k The expression of α-tubulin was detected by Western blot in each group of A549/Taxol-R cells. l : PARP has a molecular weight of 116 kDa and was cleaved into 89 and 31 kDa fragments by activated Caspase-3 in each group of A549/Taxol-R cells, the expression of cleaved-Caspase-3 was detected by Western blot in the above groups. m Recombinant Caspase-3 cleaved α-tubulin only in the combined treatment other than single treatment and cleaved-α-tubulin was an approximately 53 kDa fragment. n A schematic of the involved signal pathways that SFN metabolites and PTX disturbed microtubule dynamics and activated the intrinsic apoptosis pathway leading to apoptosis in A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Journal: Cell Death & Disease

    Article Title: Sulforaphane metabolites reduce resistance to paclitaxel via microtubule disruption

    doi: 10.1038/s41419-018-1174-9

    Figure Lengend Snippet: Combination of PTX with SFN metabolites showed a synergistic inhibition in A549/Taxol-R cells. a A549/Taxol-R cells were treated with (0, 5, 10, 15, 20, 25, 30, 35, 40 nM) combined with either 10 μM SFN-Cys or 10 μM SFN-NAC, respectively at the indicated concentrations for 24 h. Then, cell viability was determined by Cell Proliferation Assay Kit. b A549/Taxol-R cells were treated with PTX (20 nM), SFN-Cys (20 μM) or SFN-NAC (20 μM), SFN-Cys (10 μM) or SFN-NAC (10 μM) combined with PTX (10 nM), respectively for 24 h, then the cells were harvested and the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit (P: PTX 20 nM; C: SFN-Cys 20 μM; N: SFN-NAC 20 μM; PC: PTX 10 nM + SFN-Cys 10 μM; PN: PTX 10 nM + SFN-NAC 10 μM). c The histogram demonstrated the number of apoptotic cells in each group was detected by flow cytometry. d A549/Taxol-R cells was treated with both PTX (20 nM), SFN-Cys (20 μM) or SFN-NAC (20 μM), SFN-Cys (10 μM) or SFN-NAC (10 μM) combined with PTX (10 nM), respectively for 24 h, then recorded by Leica DMIRB microscope at ×40 magnification. e A549/Taxol-R cells were treated with PTX (20 nM), SFN-Cys (20 μM) or SFN-NAC (20 μM), SFN-Cys (10 μM) or SFN-NAC (10 μM) combined with PTX (10 nM) respectively for 24 h, then we harvested cells and viewed subcellular structures with a transmission electron microscope. Black arrows indicated sporadic vacuoles, white arrows indicated nucleic condensation. f The expression of Caspase-7, pro-Caspase-3 and cleaved-Caspase-3 was detected by Western blot in the groups (P: PTX 20 nM; C: SFN-Cys 20 μM; N: SFN-NAC 20 μM; PC: PTX 10 nM + SFN-Cys 10 μM; PN: PTX 10 nM + SFN-NAC 10 μM). g Cell viability was determined by Cell Proliferation Assay Kit. h Cells were harvested and the percentages of cell apoptosis were analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. i The histogram showed the number of apoptotic cells in each group. j Immunofluorescence staining of α-tubulin showed the changes of microtubule morphology treated with PTX and SFN-NAC in different groups. We also treated A549/Taxol-R cells with PTX and SFN-Cys in different groups, the results are the same as the combination of PTX and SFN-NAC (data not shown). Red: α-tubulin, Blue: DAPI-stained DNA; White arrows: normal microtubules; red arrows: the abnormal microtubules. Scale bars, 25 μm. k The expression of α-tubulin was detected by Western blot in each group of A549/Taxol-R cells. l : PARP has a molecular weight of 116 kDa and was cleaved into 89 and 31 kDa fragments by activated Caspase-3 in each group of A549/Taxol-R cells, the expression of cleaved-Caspase-3 was detected by Western blot in the above groups. m Recombinant Caspase-3 cleaved α-tubulin only in the combined treatment other than single treatment and cleaved-α-tubulin was an approximately 53 kDa fragment. n A schematic of the involved signal pathways that SFN metabolites and PTX disturbed microtubule dynamics and activated the intrinsic apoptosis pathway leading to apoptosis in A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Article Snippet: Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA).

    Techniques: Inhibition, Proliferation Assay, Flow Cytometry, Cytometry, Microscopy, Transmission Assay, Expressing, Western Blot, Immunofluorescence, Staining, Molecular Weight, Recombinant

    Establishment of PTX-resistant cell line A549/Taxol-R. a Both A549 cells and A549/Taxol-R cells treated with PTXs were analyzed for IC50 by Graphpad prism 5 software. b Both A549 and A549/Taxol-R cells were treated with gradient-concentrations of PTX for 24 h. Then, cell viability was determined by Cell Proliferation Assay Kit. Cell viability (percentage) was ratio of OD at 490 nm value of each group cells vs. OD value of control group cells. c Both A549 cells and A549/Taxol-R cells were treated with 0, 5, 10, 15, 20, 25 nM PTX and recorded by Leica DMIRB microscope at × 40 magnification for 24 h. d After treated with 20 nM PTX, both A549 and A549/Taxol-R cells were harvested and finally were viewed under TEM. Black arrow indicates nucleic fragmentation, and double black arrows indicate mitochondria. e Both A549 and A549/Taxol-R cells were treated with 0, 5, 10, 15, 20, 25 nM for 24 h, then the cells were harvested and the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. f The histogram showed the quantification of apoptosis cells of A549 and A549/Taxol-R cells. These results were from three independent experiments. g The expressions of βIII-tubulin, XIAP, Tau, Stathmin1, Hsp70 and α-tubulin were detected by Western blot in A549 cells and A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Journal: Cell Death & Disease

    Article Title: Sulforaphane metabolites reduce resistance to paclitaxel via microtubule disruption

    doi: 10.1038/s41419-018-1174-9

    Figure Lengend Snippet: Establishment of PTX-resistant cell line A549/Taxol-R. a Both A549 cells and A549/Taxol-R cells treated with PTXs were analyzed for IC50 by Graphpad prism 5 software. b Both A549 and A549/Taxol-R cells were treated with gradient-concentrations of PTX for 24 h. Then, cell viability was determined by Cell Proliferation Assay Kit. Cell viability (percentage) was ratio of OD at 490 nm value of each group cells vs. OD value of control group cells. c Both A549 cells and A549/Taxol-R cells were treated with 0, 5, 10, 15, 20, 25 nM PTX and recorded by Leica DMIRB microscope at × 40 magnification for 24 h. d After treated with 20 nM PTX, both A549 and A549/Taxol-R cells were harvested and finally were viewed under TEM. Black arrow indicates nucleic fragmentation, and double black arrows indicate mitochondria. e Both A549 and A549/Taxol-R cells were treated with 0, 5, 10, 15, 20, 25 nM for 24 h, then the cells were harvested and the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. f The histogram showed the quantification of apoptosis cells of A549 and A549/Taxol-R cells. These results were from three independent experiments. g The expressions of βIII-tubulin, XIAP, Tau, Stathmin1, Hsp70 and α-tubulin were detected by Western blot in A549 cells and A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Article Snippet: Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA).

    Techniques: Software, Proliferation Assay, Microscopy, Transmission Electron Microscopy, Flow Cytometry, Cytometry, Western Blot

    βIII-tubulin expression showed positive correlation with pathological grading in NSCLC tissues and survival analysis by GEAPIA Database. a βIII-tubulin was expressed in human NSCLC tissues associated with various histopathological grading by IHC staining. The expression of βIII-tubulin in the adjacent tissues was observed as the control ( a , e , i , m ). IHC, magnification × 40, in a – l ; magnification × 200, in e – p . b The correlation of βIII-tubulin expression with clinicopathological characteristics of lung squamous cell carcinoma patients. H scores (low: 0–4; high: 5–12). c The correlation of βIII-tubulin expression with clinicopathological characteristics of lung adenocarcinoma patients. H scores (low: 0–4; high: 5–12). d The expression of drug resistance-related protein α-tubulin and Stathmin1 in tumor patients and normal adults. LUAD (lung adenocarcinoma), LUSC (lung squamous carcinoma), T (tumor patients), N (normal adults). e The survival analysis of drug resistance-related protein α-tubulin and Hsp70 via GEAPIA Database 32 . Data were shown as * P

    Journal: Cell Death & Disease

    Article Title: Sulforaphane metabolites reduce resistance to paclitaxel via microtubule disruption

    doi: 10.1038/s41419-018-1174-9

    Figure Lengend Snippet: βIII-tubulin expression showed positive correlation with pathological grading in NSCLC tissues and survival analysis by GEAPIA Database. a βIII-tubulin was expressed in human NSCLC tissues associated with various histopathological grading by IHC staining. The expression of βIII-tubulin in the adjacent tissues was observed as the control ( a , e , i , m ). IHC, magnification × 40, in a – l ; magnification × 200, in e – p . b The correlation of βIII-tubulin expression with clinicopathological characteristics of lung squamous cell carcinoma patients. H scores (low: 0–4; high: 5–12). c The correlation of βIII-tubulin expression with clinicopathological characteristics of lung adenocarcinoma patients. H scores (low: 0–4; high: 5–12). d The expression of drug resistance-related protein α-tubulin and Stathmin1 in tumor patients and normal adults. LUAD (lung adenocarcinoma), LUSC (lung squamous carcinoma), T (tumor patients), N (normal adults). e The survival analysis of drug resistance-related protein α-tubulin and Hsp70 via GEAPIA Database 32 . Data were shown as * P

    Article Snippet: Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA).

    Techniques: Expressing, Immunohistochemistry, Staining

    SFN metabolites upregulated 26S proteasome via sustained ERK1/2 phosphorylation to degrade resistance-related proteins. a The expression of ERK1/2, phosphorylated ERK1/2 (pERK1/2) was detected by Western blot with the treatment of 0, 15, 30, 45 μM either SFN-Cys or SFN-NAC for 24 h in A549/Taxol-R cells. b The expression of ERK1/2 and pERK1/2 was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 25 μM PD98059 for 24 h in A549/Taxol-R cells. c The expression of 26 S was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 25 μM PD98059 for 24 h in A549/Taxol-R cells. d The expression of 26 S was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 0.5 μM MG132 (0.5 μM) for 24 h in A549/Taxol-R cells. e – j The expression of α-tubulin, βIII-tubulin, Stathmin1, Tau, XIAP, Hsp70 was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 0.5 μM MG132 for 24 h in A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Journal: Cell Death & Disease

    Article Title: Sulforaphane metabolites reduce resistance to paclitaxel via microtubule disruption

    doi: 10.1038/s41419-018-1174-9

    Figure Lengend Snippet: SFN metabolites upregulated 26S proteasome via sustained ERK1/2 phosphorylation to degrade resistance-related proteins. a The expression of ERK1/2, phosphorylated ERK1/2 (pERK1/2) was detected by Western blot with the treatment of 0, 15, 30, 45 μM either SFN-Cys or SFN-NAC for 24 h in A549/Taxol-R cells. b The expression of ERK1/2 and pERK1/2 was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 25 μM PD98059 for 24 h in A549/Taxol-R cells. c The expression of 26 S was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 25 μM PD98059 for 24 h in A549/Taxol-R cells. d The expression of 26 S was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 0.5 μM MG132 (0.5 μM) for 24 h in A549/Taxol-R cells. e – j The expression of α-tubulin, βIII-tubulin, Stathmin1, Tau, XIAP, Hsp70 was detected by Western blot with the treatment of either 30 μM SFN-Cys or 30 μM SFN-NAC with/without 0.5 μM MG132 for 24 h in A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Article Snippet: Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA).

    Techniques: Expressing, Western Blot

    SFN metabolites induced apoptosis via downregulating microtubule associated proteins and upregulating Hsp70 in A549/Taxol-R cells. a A549/Taxol-R cells were treated with either SFN-Cys or SFN-NAC (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) at the indicated concentrations for 24 h. Then, cell viability was determined by Cell Proliferation Assay Kit. b A549/Taxol-R cells were treated with either SFN-Cys or SFN-NAC (0, 15, 30, 45 μM) and recorded by Leica DMIRB microscope at × 40 magnification for 24 h. c After treated with either SFN-Cys or SFN-NAC (30 μM) for 24 h, A549/Taxol-R cells were harvested and were viewed with a transmission electron microscope. Black arrow indicates sporadic vacuoles, double lack arrows indicate nucleic condensation like a flower ring, arrow head indicates karyopyknosis, double arrow heads indicate apoptotic body. d A549/Taxol-R cells were treated with either SFN-Cys or SFN-NAC (0, 15, 20, 30 μM) for 24 h, the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. e – j The expression of α-tubulin, βIII-tubulin, Stathmin1, Tau, XIAP, Hsp70 was detected by Western blot with the treatment of either 0, 15, 30, 45 μM SFN-Cys or SFN-NAC in bothA549 and A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Journal: Cell Death & Disease

    Article Title: Sulforaphane metabolites reduce resistance to paclitaxel via microtubule disruption

    doi: 10.1038/s41419-018-1174-9

    Figure Lengend Snippet: SFN metabolites induced apoptosis via downregulating microtubule associated proteins and upregulating Hsp70 in A549/Taxol-R cells. a A549/Taxol-R cells were treated with either SFN-Cys or SFN-NAC (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) at the indicated concentrations for 24 h. Then, cell viability was determined by Cell Proliferation Assay Kit. b A549/Taxol-R cells were treated with either SFN-Cys or SFN-NAC (0, 15, 30, 45 μM) and recorded by Leica DMIRB microscope at × 40 magnification for 24 h. c After treated with either SFN-Cys or SFN-NAC (30 μM) for 24 h, A549/Taxol-R cells were harvested and were viewed with a transmission electron microscope. Black arrow indicates sporadic vacuoles, double lack arrows indicate nucleic condensation like a flower ring, arrow head indicates karyopyknosis, double arrow heads indicate apoptotic body. d A549/Taxol-R cells were treated with either SFN-Cys or SFN-NAC (0, 15, 20, 30 μM) for 24 h, the percentage of cell apoptosis was analyzed by flow cytometry via Annexin V-FITC/PI Apoptosis Detection Kit. e – j The expression of α-tubulin, βIII-tubulin, Stathmin1, Tau, XIAP, Hsp70 was detected by Western blot with the treatment of either 0, 15, 30, 45 μM SFN-Cys or SFN-NAC in bothA549 and A549/Taxol-R cells. Data were shown as means ± SD from three separate experiments. * P

    Article Snippet: Anti-Caspase-3, anti-β-actin, anti-α-tubulin, anti-Tau and protein A/G PLUS agarose were purchased from Santa Cruz Biotechnology (USA).

    Techniques: Proliferation Assay, Microscopy, Transmission Assay, Flow Cytometry, Cytometry, Expressing, Western Blot

    PGE 2 promotes EGFR nuclear translocation via EP3 receptor ( A ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 exposed for 60 min to 1 μM EP2, EP3 and EP4 agonists. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction, respectively. ( B ) Confocal analysis of EGFR localization in A549 exposed to EP3 agonist for 60 min. EGFR was stained in green, DAPI (blue) was used to counterstain the nuclei. Confocal images were captured in the middle section of the nuclei using 63x objective. Scale bars, 20 μm. Boxed areas are shown in detail in the inset. ( C ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 cells pretreated with or without EP3 antagonist (L798-106; 1 μM) for 30 min before challenging with 1μM PGE 2 for 60 min. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. *** p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: PGE 2 promotes EGFR nuclear translocation via EP3 receptor ( A ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 exposed for 60 min to 1 μM EP2, EP3 and EP4 agonists. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction, respectively. ( B ) Confocal analysis of EGFR localization in A549 exposed to EP3 agonist for 60 min. EGFR was stained in green, DAPI (blue) was used to counterstain the nuclei. Confocal images were captured in the middle section of the nuclei using 63x objective. Scale bars, 20 μm. Boxed areas are shown in detail in the inset. ( C ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 cells pretreated with or without EP3 antagonist (L798-106; 1 μM) for 30 min before challenging with 1μM PGE 2 for 60 min. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. *** p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Expressing, Staining

    SRC family kinases play a pivotal role in PGE 2 induced EGFR nuclear translocation Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 ( A , B ) and GLC82 ( C ) exposed for 60 min to 1 μM PGE 2 with or without 10 μM PP1 or SU6656. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. ** p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: SRC family kinases play a pivotal role in PGE 2 induced EGFR nuclear translocation Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 ( A , B ) and GLC82 ( C ) exposed for 60 min to 1 μM PGE 2 with or without 10 μM PP1 or SU6656. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. ** p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Expressing

    EGFR nuclear translocation in response to PGE 2 does not involve PKA, AKT and PKC ( A , B , C ) A549 and GLC82 cells were starved overnight and then treated with 1 μM H89 or 10 μM LY294002 or 10 μM Go6983 for 30 min before challenge with 1 μM PGE 2 for 60 min. Immunoblotting analysis of EGFR expression on cytoplasmic and nuclear fractions was then performed. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. ** p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: EGFR nuclear translocation in response to PGE 2 does not involve PKA, AKT and PKC ( A , B , C ) A549 and GLC82 cells were starved overnight and then treated with 1 μM H89 or 10 μM LY294002 or 10 μM Go6983 for 30 min before challenge with 1 μM PGE 2 for 60 min. Immunoblotting analysis of EGFR expression on cytoplasmic and nuclear fractions was then performed. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. ** p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Expressing

    PGE 2 acts via shedding of EGF-like ligands to promote EGFR nuclear translocation A549 were starved overnight and then pre-treated with 10 μM or 25 μM GM6001 before challenge with 1 μM PGE 2 for 60 min. DMSO, matching the solvent concentration of 25 μM GM6001, was used as a control. ( A ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 treated as described above. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. *** p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: PGE 2 acts via shedding of EGF-like ligands to promote EGFR nuclear translocation A549 were starved overnight and then pre-treated with 10 μM or 25 μM GM6001 before challenge with 1 μM PGE 2 for 60 min. DMSO, matching the solvent concentration of 25 μM GM6001, was used as a control. ( A ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 treated as described above. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. *** p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Concentration Assay, Expressing

    NSCLC cell models to study PGE 2 -induced EGFR nuclear translocation ( A ) Immunoblotting analysis of EGFR expression in A549 wild type cells and two clones knockout for EGFR, generated by CRISPR/Cas9 (EGFR −/− #1, #2). Actin was used as loading control. ( B , C ) EGFR knockout cells were transiently transfected with Vector or EGFR-WT or EGFR mutated in NLS (NLSm12 or dNLS) plasmids for 48 h. Then EGFR nuclear translocation in response to 25 ng/ml EGF for 10 min (B) or 1 μM PGE 2 for 60 min (C) was analyzed by immunoblotting upon cell fractionation. Parental cells were included as a control. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. ( D ) Immunoblotting analysis of EGFR phosphorylation on tyrosine 1068 and AKT on serine 473, upon EGF treatment in parental and EGFR knockout cells expressing Vector, EGFR-WT and NLS mutant plasmids. ( E ) Immunoblotting quantification of pEGFR Tyr 1068, normalized to EGFR and pAKT Ser 473, normalized to AKT, were expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. * p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: NSCLC cell models to study PGE 2 -induced EGFR nuclear translocation ( A ) Immunoblotting analysis of EGFR expression in A549 wild type cells and two clones knockout for EGFR, generated by CRISPR/Cas9 (EGFR −/− #1, #2). Actin was used as loading control. ( B , C ) EGFR knockout cells were transiently transfected with Vector or EGFR-WT or EGFR mutated in NLS (NLSm12 or dNLS) plasmids for 48 h. Then EGFR nuclear translocation in response to 25 ng/ml EGF for 10 min (B) or 1 μM PGE 2 for 60 min (C) was analyzed by immunoblotting upon cell fractionation. Parental cells were included as a control. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. ( D ) Immunoblotting analysis of EGFR phosphorylation on tyrosine 1068 and AKT on serine 473, upon EGF treatment in parental and EGFR knockout cells expressing Vector, EGFR-WT and NLS mutant plasmids. ( E ) Immunoblotting quantification of pEGFR Tyr 1068, normalized to EGFR and pAKT Ser 473, normalized to AKT, were expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. * p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Expressing, Clone Assay, Knock-Out, Generated, CRISPR, Transfection, Plasmid Preparation, Cell Fractionation, Mutagenesis

    EGFR kinase domain is necessary for its nuclear translocation ( A ) Immunoblotting analysis of EGFR phosphorylation on Tyr1068, and AKT phosphorylation on Ser473 in A549 exposed to 1 μM PGE 2 for 5–60 min. GAPDH was used as loading control ( B , C ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 exposed for 10 min to 25 ng/ml EGF (B), or 60 min to 1 μM PGE2 (C), with or without pre-incubation with AG1478 (10 μM) for 30 min. ( D ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in GLC82 exposed for 10 min to 25 ng/ml EGF, or 60 min to 1μM PGE 2 , with or without pre-incubation with 10 μM AG1478 for 30 min. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. ** p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: EGFR kinase domain is necessary for its nuclear translocation ( A ) Immunoblotting analysis of EGFR phosphorylation on Tyr1068, and AKT phosphorylation on Ser473 in A549 exposed to 1 μM PGE 2 for 5–60 min. GAPDH was used as loading control ( B , C ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in A549 exposed for 10 min to 25 ng/ml EGF (B), or 60 min to 1 μM PGE2 (C), with or without pre-incubation with AG1478 (10 μM) for 30 min. ( D ) Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in GLC82 exposed for 10 min to 25 ng/ml EGF, or 60 min to 1μM PGE 2 , with or without pre-incubation with 10 μM AG1478 for 30 min. Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. ** p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Expressing, Incubation

    PGE 2 induces EGFR nuclear translocation Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in overnight starved A549 ( A , B , C ) and GLC82 ( D , E , F ). Cells were exposed for 10–120 min to 25 ng/ml EGF (A, D) or 1 μM PGE 2 (B, E). Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. * p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: PGE 2 induces EGFR nuclear translocation Immunoblotting analysis of EGFR expression in cytosolic and nuclear fraction in overnight starved A549 ( A , B , C ) and GLC82 ( D , E , F ). Cells were exposed for 10–120 min to 25 ng/ml EGF (A, D) or 1 μM PGE 2 (B, E). Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. * p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Expressing

    EGFR ligands mediate PGE 2 -dependent EGFR nuclear translocation ( A ) qRT-PCR analysis of basal mRNA expression for EGFR ligands in A549. Results are presented as mean of Ct values ± SEM of two independent experiments. ( B ) A549 cells were starved overnight and then treated with 1 μM PGE 2 for 2, 4, 8, 12, 18 and 24 h. RNA was isolated and analyzed by qRT-PCR for EGFR ligands. The data are presented as mean of fold change ± SEM of three independent experiments, relative to non-treated cells (Control), which were assigned to 1. ( C ) mRNA expression analysis of EGFR ligands by qRT-PCR in A549 silenced for 48 h for AREG, EREG, TGF-alpha and HB-EGF with two different oligos (siRNA A and B) (left panel). EGFR expression analysis by immunoblotting of cytosolic and nuclear fraction in A549 silenced for EGFR ligands (right panel). Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. *** p

    Journal: Oncotarget

    Article Title: PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

    doi: 10.18632/oncotarget.16116

    Figure Lengend Snippet: EGFR ligands mediate PGE 2 -dependent EGFR nuclear translocation ( A ) qRT-PCR analysis of basal mRNA expression for EGFR ligands in A549. Results are presented as mean of Ct values ± SEM of two independent experiments. ( B ) A549 cells were starved overnight and then treated with 1 μM PGE 2 for 2, 4, 8, 12, 18 and 24 h. RNA was isolated and analyzed by qRT-PCR for EGFR ligands. The data are presented as mean of fold change ± SEM of three independent experiments, relative to non-treated cells (Control), which were assigned to 1. ( C ) mRNA expression analysis of EGFR ligands by qRT-PCR in A549 silenced for 48 h for AREG, EREG, TGF-alpha and HB-EGF with two different oligos (siRNA A and B) (left panel). EGFR expression analysis by immunoblotting of cytosolic and nuclear fraction in A549 silenced for EGFR ligands (right panel). Tubulin and Lamin A were used as loading control for cytosolic and nuclear fraction respectively. Immunoblotting quantification was expressed in A.D.U. (arbitrary density unit) and as mean ± SEM. *** p

    Article Snippet: Anti-Tubulin and anti-EP3 receptor antibodies were purchased from Santa Cruz (Heidelberg, Germany).

    Techniques: Translocation Assay, Quantitative RT-PCR, Expressing, Isolation

    Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). β-Tubulin (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).

    Journal: PLoS ONE

    Article Title: The effect of “NutramilTM Complex,” food for special medical purpose, on breast and prostate carcinoma cells

    doi: 10.1371/journal.pone.0192860

    Figure Lengend Snippet: Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells. MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, Nutramil TM Complex, at 4% concentration; NC-CC, Nutramil TM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 μM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). β-Tubulin (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).

    Article Snippet: Subsequently, the immobilized proteins were incubated with the appropriate primary antibody: cytochrome c (#11940), Smac/Diablo (#2954), HtrA2/Omi (#9745) and β-Tubulin (#2128) (Cell Signaling Technology, MA, USA).

    Techniques: Expressing, Concentration Assay, Positive Control, Lysis, Protease Inhibitor, Incubation, Western Blot

    C-terminal tubulin tail mutants increase microtubule dynamics and modulate cell mechanic-dependent activities. (a-d) HeLa cells were transfected with TUBA1A constructs and plated on 50kPa hydrogel. (a) Representative alignment of microtubule in cells. (b) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin in cells. (c) Proliferation rate of cells. (d) Cell velocity (left) and speed (right) of cells. In all the panels n > 50 cells from 3 independent experiments were analyzed. *P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: C-terminal tubulin tail mutants increase microtubule dynamics and modulate cell mechanic-dependent activities. (a-d) HeLa cells were transfected with TUBA1A constructs and plated on 50kPa hydrogel. (a) Representative alignment of microtubule in cells. (b) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin in cells. (c) Proliferation rate of cells. (d) Cell velocity (left) and speed (right) of cells. In all the panels n > 50 cells from 3 independent experiments were analyzed. *P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Transfection, Construct, Diffusion-based Assay

    TTLL4 force microtubule glutamylation to adjust cell mechanics and sustain cell mechanic-dependent activities. (a-f , h) HeLa cells plated on 50 kPa hydrogel were transfected with the indicated siRNA. (a) Immunoblot and quantification of Glu-Tubulin in cells. (b) Representative confocal images (left) and quantification (right) of Glu-Tubulin and Tubulin. Scale bar=10 µm. (c) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP. n > 500 comets. Scale bar=1 µm. (d) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin. (e) Apparent Young’s moduli obtained by AFM analysis. Bars represent the median. (f) Representative heat map (left) and quantification (right) showing contractile forces generate by cells. (g) Representative confocal images (left) and quantification (right) of circularity index. Scale bar=10 µm. In all the panels n > 20 cells from 3 independent experiments were analyzed. *P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: TTLL4 force microtubule glutamylation to adjust cell mechanics and sustain cell mechanic-dependent activities. (a-f , h) HeLa cells plated on 50 kPa hydrogel were transfected with the indicated siRNA. (a) Immunoblot and quantification of Glu-Tubulin in cells. (b) Representative confocal images (left) and quantification (right) of Glu-Tubulin and Tubulin. Scale bar=10 µm. (c) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP. n > 500 comets. Scale bar=1 µm. (d) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin. (e) Apparent Young’s moduli obtained by AFM analysis. Bars represent the median. (f) Representative heat map (left) and quantification (right) showing contractile forces generate by cells. (g) Representative confocal images (left) and quantification (right) of circularity index. Scale bar=10 µm. In all the panels n > 20 cells from 3 independent experiments were analyzed. *P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Transfection, Diffusion-based Assay

    TTLL5 orTTLL9 cell depletion decreased MT glutamylation, reorganize the microtubule lattice and affect cell mechanic-dependent cell functions (a-l) HeLa cells were transfected with the indicated siRNA (control, siCtrl; TTLL and CCP, siRNA single, _s1, s2, s3) for 48h and plated on 50kPa hydrogel. (a , b) Immunoblot of Glu-Tubulin in cells. (c , e) Representative confocal images (left) and quantification (right; n > 50 cells from 3 independent experiments) of Glu-Tubulin and Tubulin in cells. Scale bar=10 µm. At least 10 cells per condition from n=3 independent experiments. (d, f) Representative alignment of microtubule in cells. (g , h) Quantification of circularity index of cells. (i-j) Proliferation rate of cells. (k-l) Cell velocity (left) and speed (right) of cells. In all the panels n > 50 cells from 3 independent experiments were analyzed. *P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: TTLL5 orTTLL9 cell depletion decreased MT glutamylation, reorganize the microtubule lattice and affect cell mechanic-dependent cell functions (a-l) HeLa cells were transfected with the indicated siRNA (control, siCtrl; TTLL and CCP, siRNA single, _s1, s2, s3) for 48h and plated on 50kPa hydrogel. (a , b) Immunoblot of Glu-Tubulin in cells. (c , e) Representative confocal images (left) and quantification (right; n > 50 cells from 3 independent experiments) of Glu-Tubulin and Tubulin in cells. Scale bar=10 µm. At least 10 cells per condition from n=3 independent experiments. (d, f) Representative alignment of microtubule in cells. (g , h) Quantification of circularity index of cells. (i-j) Proliferation rate of cells. (k-l) Cell velocity (left) and speed (right) of cells. In all the panels n > 50 cells from 3 independent experiments were analyzed. *P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Transfection

    Microtubules glutamylation is orchestrated by TTLL4 and CCP5 to adjust cell mechanics and sustain cell mechanic-dependent activities. (a-j) HeLa cells were transfected with the indicated siRNA (control, siCtrl; TTLL and CCP, siRNA single, _s1, s2, s3) for 48h. (a) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP in cells plated on 1kPa hydrogel. n > 500 comets. Scale bar=1 µm. (b) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin in cells plated on 1kPa hydrogel (n > 45 cells). (c) Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel. (d) Representative confocal images (left) and quantification (right) of circularity index of cells plated on 1kPa hydrogel. Scale bar=10 µm. (e-f) Proliferation rate of cells plated on 50kPa (e) or 1kPa (f) hydrogel. (g-h) Measurement of cell adhesion on cells plated on 50kPa (g) or 1kPa (h) hydrogel. (i-j) Cell velocity (left) and speed (right) of cells plated on 50kPa (i) or 1kPa (j) hydrogel. In all the panels n > 50 cells from 3 independent experiments were analyzed. *P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: Microtubules glutamylation is orchestrated by TTLL4 and CCP5 to adjust cell mechanics and sustain cell mechanic-dependent activities. (a-j) HeLa cells were transfected with the indicated siRNA (control, siCtrl; TTLL and CCP, siRNA single, _s1, s2, s3) for 48h. (a) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP in cells plated on 1kPa hydrogel. n > 500 comets. Scale bar=1 µm. (b) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin in cells plated on 1kPa hydrogel (n > 45 cells). (c) Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel. (d) Representative confocal images (left) and quantification (right) of circularity index of cells plated on 1kPa hydrogel. Scale bar=10 µm. (e-f) Proliferation rate of cells plated on 50kPa (e) or 1kPa (f) hydrogel. (g-h) Measurement of cell adhesion on cells plated on 50kPa (g) or 1kPa (h) hydrogel. (i-j) Cell velocity (left) and speed (right) of cells plated on 50kPa (i) or 1kPa (j) hydrogel. In all the panels n > 50 cells from 3 independent experiments were analyzed. *P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Transfection, Diffusion-based Assay

    Mechanical cues increase microtubules glutamylation and alter microtubules dynamics to force cell mechanics. (a) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of endogenous Tubulin labeled with Oregon Green™ 488 Taxol, Bis-Acetate in HeLa cells plated on different stiffness hydrogel (1, 12, 50 kPa) or plastic (n > 45 cells). (b) Representative heat map (left) and quantification (right; n > 15 cells from n=3 independent experiments) showing contractile forces generate by cells plated on 12kPa hydrogel and treated with Nocodazole or Taxol. (c) Immunoblot and quantification (n=3 independent experiments) of Glu-Tubulin in HeLa cells plated on 1 kPa hydrogel and after shear stress for the indicated times. Hsp90 was used as a loading control. (d , e) Immunoblot of Glu-Tubulin in MDA-MB-468 cells (d) and primary pulmonary arterial smooth muscle cells (PASMCs; e) plated on the indicated substrate. (f , h) Representative confocal images of Tubulin and Glu-Tubulin localization in HeLa cells plated on different stiffness hydrogel (1, 12, 50 kPa) or plated on 1kPa hydrogel and after osmotic stress for the indicated times. Nuclei were stained with DAPI (Blue) on the MERGE image. Quantification (right) of Glu-Tubulin intensity in the different condition. At least 50 cells per condition. Scale bar=10 µm. (g) Representative alignment of microtubule in HeLa cells plated on different stiffness hydrogel (1, 12, 50 kPa). At least 10 cells per condition. n=3 independent experiments; **P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: Mechanical cues increase microtubules glutamylation and alter microtubules dynamics to force cell mechanics. (a) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of endogenous Tubulin labeled with Oregon Green™ 488 Taxol, Bis-Acetate in HeLa cells plated on different stiffness hydrogel (1, 12, 50 kPa) or plastic (n > 45 cells). (b) Representative heat map (left) and quantification (right; n > 15 cells from n=3 independent experiments) showing contractile forces generate by cells plated on 12kPa hydrogel and treated with Nocodazole or Taxol. (c) Immunoblot and quantification (n=3 independent experiments) of Glu-Tubulin in HeLa cells plated on 1 kPa hydrogel and after shear stress for the indicated times. Hsp90 was used as a loading control. (d , e) Immunoblot of Glu-Tubulin in MDA-MB-468 cells (d) and primary pulmonary arterial smooth muscle cells (PASMCs; e) plated on the indicated substrate. (f , h) Representative confocal images of Tubulin and Glu-Tubulin localization in HeLa cells plated on different stiffness hydrogel (1, 12, 50 kPa) or plated on 1kPa hydrogel and after osmotic stress for the indicated times. Nuclei were stained with DAPI (Blue) on the MERGE image. Quantification (right) of Glu-Tubulin intensity in the different condition. At least 50 cells per condition. Scale bar=10 µm. (g) Representative alignment of microtubule in HeLa cells plated on different stiffness hydrogel (1, 12, 50 kPa). At least 10 cells per condition. n=3 independent experiments; **P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Diffusion-based Assay, Labeling, Multiple Displacement Amplification, Staining

    Mechanical cues force microtubules glutamylation to stabilize the microtubule lattice. (a) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin (n > 45 cells) (b) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP in cells plated on the indicated substrate. n > 500 comets. Scale bar=1 µm. (c-d) Immunoblot and quantification of Glu-Tubulin in cells plated on the indicated substrate (c) or after hypo-osmotic shock ( d). (e-f) Representative STED images of Tubulin and Glu-Tubulin localization (e) and representative alignment of microtubule (f) in cells plated on 1 or 50 kPa hydrogel (n > 10). Scale bar=10 µm; for the inset, scale bar=1 µm.; *P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: Mechanical cues force microtubules glutamylation to stabilize the microtubule lattice. (a) Representative FRAP curves (left) and quantification of diffusion rate (t ½) and mobile fraction (right) of GFP-Tubulin (n > 45 cells) (b) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP in cells plated on the indicated substrate. n > 500 comets. Scale bar=1 µm. (c-d) Immunoblot and quantification of Glu-Tubulin in cells plated on the indicated substrate (c) or after hypo-osmotic shock ( d). (e-f) Representative STED images of Tubulin and Glu-Tubulin localization (e) and representative alignment of microtubule (f) in cells plated on 1 or 50 kPa hydrogel (n > 10). Scale bar=10 µm; for the inset, scale bar=1 µm.; *P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Diffusion-based Assay

    Microtubule glutamylation is sufficient to adjust cell mechanics and sustain cell mechanic-dependent activities. (a) Schematic representation of TUBA1A structure in wild type and mutant. (b-g) HeLa cells were transfected with TUBA1A constructs. (b) Immunoblot and quantification of Glu-Tubulin in cells. (c) Representative confocal images (left) and quantification (right) of Glu-Tubulin and Tubulin. Scale bar=10 µm. *: Transfected cell (d) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP. n > 500 comets. Scale bar=1 µm. (e) Apparent Young’s moduli obtained by AFM analysis. Bars represent the median. (f) Representative heat map (left) and quantification (right) showing contractile forces generate by cells. (g) Representative confocal images (left) and quantification (right) of circularity index. Scale bar=10 µm. In all the panels n > 50 cells from 3 independent experiments were analyzed. **P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: Microtubule glutamylation is sufficient to adjust cell mechanics and sustain cell mechanic-dependent activities. (a) Schematic representation of TUBA1A structure in wild type and mutant. (b-g) HeLa cells were transfected with TUBA1A constructs. (b) Immunoblot and quantification of Glu-Tubulin in cells. (c) Representative confocal images (left) and quantification (right) of Glu-Tubulin and Tubulin. Scale bar=10 µm. *: Transfected cell (d) Representative kymographs (left) and growth rates quantification (right) of EB1-GFP. n > 500 comets. Scale bar=1 µm. (e) Apparent Young’s moduli obtained by AFM analysis. Bars represent the median. (f) Representative heat map (left) and quantification (right) showing contractile forces generate by cells. (g) Representative confocal images (left) and quantification (right) of circularity index. Scale bar=10 µm. In all the panels n > 50 cells from 3 independent experiments were analyzed. **P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Mutagenesis, Transfection, Construct

    Balanced microtubules glutamylation by TTL4 and CCP5 organize the microtubule lattice. (a-i) HeLa cells were transfected with the indicated siRNA (control, siCtrl; TTLL and CCP, siRNA smarpool or siRNA single, _s1, s2, s3) for 48h. (a , c-d) As demonstrated by RT-qPCR, effective siRNA knockdown was achieved in Hela cells. For each gene transcript, mean expression in control groups (siCtrl) were assigned a fold change of 1, to which relevant samples (transfected with a siRNA specific to that gene) were compared . (b , e-f) Immunoblot (b , e-f) and quantification (b) of Glu-Tubulin in cells. (g, i) Representative alignment of microtubule in cells in plated on 50 kPa (g) or 1kPa (i) hydrogels. (h) Representative confocal images (left) and quantification (right; n > 50 cells from 3 independent experiments) of Glu-Tubulin and Tubulin in cells plated on 1kPa hydrogel. Scale bar=10 µm. At least 10 cells per condition from n=3 independent experiments; *P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: Balanced microtubules glutamylation by TTL4 and CCP5 organize the microtubule lattice. (a-i) HeLa cells were transfected with the indicated siRNA (control, siCtrl; TTLL and CCP, siRNA smarpool or siRNA single, _s1, s2, s3) for 48h. (a , c-d) As demonstrated by RT-qPCR, effective siRNA knockdown was achieved in Hela cells. For each gene transcript, mean expression in control groups (siCtrl) were assigned a fold change of 1, to which relevant samples (transfected with a siRNA specific to that gene) were compared . (b , e-f) Immunoblot (b , e-f) and quantification (b) of Glu-Tubulin in cells. (g, i) Representative alignment of microtubule in cells in plated on 50 kPa (g) or 1kPa (i) hydrogels. (h) Representative confocal images (left) and quantification (right; n > 50 cells from 3 independent experiments) of Glu-Tubulin and Tubulin in cells plated on 1kPa hydrogel. Scale bar=10 µm. At least 10 cells per condition from n=3 independent experiments; *P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Transfection, Quantitative RT-PCR, Expressing

    Mechanoactivation of GLS-dependent glutamine catabolism sustain microtubules glutamylation under mechanical stresses. (a-b, d-e) HeLa cells plated on the indicated substrate and treated with CB839 or BPTES. (a) Immunoblot and quantification (n=3 independent experiments) of Glu-Tubulin in cells. (b) Immunoblot and quantification (n=3) of Glu-Tubulin in HeLa cells after osmotic stress for the indicated times. Hsp90 was used as a loading control. (c) Immunoblot and quantification (n=3) of Glu-Tubulin in HeLa cells transfected with the siRNA GLS in presence of glutamate for 24h. (d) Representative confocal images (left) and quantification (right; n > 50 cells from n=3 independent experiments) of Glu-Tubulin and Tubulin in presence of glutamate for 24h. Nuclei were stained with DAPI (Blue) on the MERGE image.Scale bar=10 µm. At least 50 cells per condition from 3 independent experiments. Scale bar=10 µm. (e) Representative alignment of microtubule in cells in presence of glutamate for 24h. At least 10 cells per condition from n=3 independent experiments; *P

    Journal: bioRxiv

    Article Title: Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

    doi: 10.1101/2020.03.10.985036

    Figure Lengend Snippet: Mechanoactivation of GLS-dependent glutamine catabolism sustain microtubules glutamylation under mechanical stresses. (a-b, d-e) HeLa cells plated on the indicated substrate and treated with CB839 or BPTES. (a) Immunoblot and quantification (n=3 independent experiments) of Glu-Tubulin in cells. (b) Immunoblot and quantification (n=3) of Glu-Tubulin in HeLa cells after osmotic stress for the indicated times. Hsp90 was used as a loading control. (c) Immunoblot and quantification (n=3) of Glu-Tubulin in HeLa cells transfected with the siRNA GLS in presence of glutamate for 24h. (d) Representative confocal images (left) and quantification (right; n > 50 cells from n=3 independent experiments) of Glu-Tubulin and Tubulin in presence of glutamate for 24h. Nuclei were stained with DAPI (Blue) on the MERGE image.Scale bar=10 µm. At least 50 cells per condition from 3 independent experiments. Scale bar=10 µm. (e) Representative alignment of microtubule in cells in presence of glutamate for 24h. At least 10 cells per condition from n=3 independent experiments; *P

    Article Snippet: The following commercially available antibodies were used for western blotting and immunofluorescence: - mouse monoclonal antibodies against Glu-Tubulin (CliniSciences, AG-20B-0020-C100), Tubulin (Santa Cruz Biotechnology, sc-398937), Hsp90 (Santa Cruz Biotechnology, sc-69703), Hsp60 (Santa Cruz Biotechnology, sc-271215); - rabbit polyclonal antibodies against Tubulin (Sigma-Aldrich, SAB4500087), TTLL4 (Bio-Techne, NBP1-81535), CCP5 (Abcam, ab170541), GLS (Abcam, ab93434).

    Techniques: Transfection, Staining

    PGI 2 mimics iloprost-induced contractile apparatus and connexin 43 protein expression. ( A ) Effect of PGI 2 on PKA activity. Human uSMCs were treated for 20 min with PGI 2 or iloprost at the concentrations indicated and subjected to in vitro PKA assay. The phosphorylated kemptide gel is shown. ( B and C ) Effect of PGI 2 on contractile apparatus protein and connexin 43 expression. ( B ) Human uSMCs were treated with PGI 2 every 1.5 h up to 6 h or with a single treatment of iloprost for 6 h at the concentrations indicated. Western blots were performed with antibodies against SM2-MHC, calponin, β-tubulin, and GAPDH. ( C ) uSMCs were subjected to a single treatment of 0.6 μmol/l PGI 2 for the indicated times. Western blot analysis was performed with antibodies against SM2-MHC, h-caldesmon, connexin 43, and β-tubulin as a loading control.

    Journal: The Journal of Clinical Investigation

    Article Title: Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

    doi: 10.1172/JCI33800

    Figure Lengend Snippet: PGI 2 mimics iloprost-induced contractile apparatus and connexin 43 protein expression. ( A ) Effect of PGI 2 on PKA activity. Human uSMCs were treated for 20 min with PGI 2 or iloprost at the concentrations indicated and subjected to in vitro PKA assay. The phosphorylated kemptide gel is shown. ( B and C ) Effect of PGI 2 on contractile apparatus protein and connexin 43 expression. ( B ) Human uSMCs were treated with PGI 2 every 1.5 h up to 6 h or with a single treatment of iloprost for 6 h at the concentrations indicated. Western blots were performed with antibodies against SM2-MHC, calponin, β-tubulin, and GAPDH. ( C ) uSMCs were subjected to a single treatment of 0.6 μmol/l PGI 2 for the indicated times. Western blot analysis was performed with antibodies against SM2-MHC, h-caldesmon, connexin 43, and β-tubulin as a loading control.

    Article Snippet: Equal amounts of protein (10–20 μg) from each lysate were subjected to Western blot analysis as described previously ( ) using primary antibodies against α-SMA, calponin, or h-caldesmon (Sigma-Aldrich); SM2-MHC (Seikagaku America); connexin 43 (Cell Signaling); PKA Cα, PKA Cβ, or β-tubulin (Santa Cruz Biotechnology Inc.); or GAPDH (AbCam).

    Techniques: Expressing, Activity Assay, In Vitro, Protein Kinase A Assay, Western Blot

    Cicaprost increases PKA activity as well as contractile protein and connexin 43 expression. ( A ) Human uSMCs were treated with vehicle or with the indicated concentrations of cicaprost for 20 min. Cell lysates were analyzed for PKA activity as described in Methods. Densitometric quantitation of fluorescence units of phosphorylated kemptide from the gel shown are expressed as fold induction. ( B ) Human uSMCs were treated with 2.5 nmol/l cicaprost or iloprost for the indicated times, and cells were harvested and analyzed for PKA activity. The phosphorylated kemptide gel is shown. ( C ) Cell lysates from human uSMCs were treated with the indicated concentrations of cicaprost for 8 h and were subjected to Western blot analysis with antibodies to α-SMA, h-caldesmon, SM2-MHC, and β-tubulin as a loading control. ( D and E ) Cells were treated with 2.5 nmol/l cicaprost for the indicated times or 2.5 nmol/l iloprost for 8 h, and Western blots were performed using antibodies against ( D ) α-SMA and calponin or ( E ) SM2-MHC. GAPDH and β-tubulin served as loading controls. ( F ) uSMCs were treated with 2.5 nmol/l cicaprost, and Western blot analysis was performed for connexin 43 and β-tubulin as a loading control.

    Journal: The Journal of Clinical Investigation

    Article Title: Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

    doi: 10.1172/JCI33800

    Figure Lengend Snippet: Cicaprost increases PKA activity as well as contractile protein and connexin 43 expression. ( A ) Human uSMCs were treated with vehicle or with the indicated concentrations of cicaprost for 20 min. Cell lysates were analyzed for PKA activity as described in Methods. Densitometric quantitation of fluorescence units of phosphorylated kemptide from the gel shown are expressed as fold induction. ( B ) Human uSMCs were treated with 2.5 nmol/l cicaprost or iloprost for the indicated times, and cells were harvested and analyzed for PKA activity. The phosphorylated kemptide gel is shown. ( C ) Cell lysates from human uSMCs were treated with the indicated concentrations of cicaprost for 8 h and were subjected to Western blot analysis with antibodies to α-SMA, h-caldesmon, SM2-MHC, and β-tubulin as a loading control. ( D and E ) Cells were treated with 2.5 nmol/l cicaprost for the indicated times or 2.5 nmol/l iloprost for 8 h, and Western blots were performed using antibodies against ( D ) α-SMA and calponin or ( E ) SM2-MHC. GAPDH and β-tubulin served as loading controls. ( F ) uSMCs were treated with 2.5 nmol/l cicaprost, and Western blot analysis was performed for connexin 43 and β-tubulin as a loading control.

    Article Snippet: Equal amounts of protein (10–20 μg) from each lysate were subjected to Western blot analysis as described previously ( ) using primary antibodies against α-SMA, calponin, or h-caldesmon (Sigma-Aldrich); SM2-MHC (Seikagaku America); connexin 43 (Cell Signaling); PKA Cα, PKA Cβ, or β-tubulin (Santa Cruz Biotechnology Inc.); or GAPDH (AbCam).

    Techniques: Activity Assay, Expressing, Quantitation Assay, Fluorescence, Western Blot

    RO3244794 inhibits contractile protein expression induced by iloprost, but not by 8-Br-cAMP. ( A ) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with vehicle or 2.5 nmol/l iloprost for 20 min. Cell lysates were analyzed for PKA activity using a nonradioactive in vitro PKA assay. A representative experiment is shown. ( B and C ) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with or without 2.5 nmol/l iloprost for 8 h. Cell lysates were prepared and subjected to Western blot analysis with antibodies against ( B ) h-caldesmon and ( C ) SM2-MHC. Lanes were run on the same gel but were noncontiguous (lines). ( D ) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with 0.5 μM 8-Br-cAMP for 8 h. Cell lysates were subjected to Western blot analysis with antibodies against h-caldesmon and β-tubulin.

    Journal: The Journal of Clinical Investigation

    Article Title: Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

    doi: 10.1172/JCI33800

    Figure Lengend Snippet: RO3244794 inhibits contractile protein expression induced by iloprost, but not by 8-Br-cAMP. ( A ) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with vehicle or 2.5 nmol/l iloprost for 20 min. Cell lysates were analyzed for PKA activity using a nonradioactive in vitro PKA assay. A representative experiment is shown. ( B and C ) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with or without 2.5 nmol/l iloprost for 8 h. Cell lysates were prepared and subjected to Western blot analysis with antibodies against ( B ) h-caldesmon and ( C ) SM2-MHC. Lanes were run on the same gel but were noncontiguous (lines). ( D ) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with 0.5 μM 8-Br-cAMP for 8 h. Cell lysates were subjected to Western blot analysis with antibodies against h-caldesmon and β-tubulin.

    Article Snippet: Equal amounts of protein (10–20 μg) from each lysate were subjected to Western blot analysis as described previously ( ) using primary antibodies against α-SMA, calponin, or h-caldesmon (Sigma-Aldrich); SM2-MHC (Seikagaku America); connexin 43 (Cell Signaling); PKA Cα, PKA Cβ, or β-tubulin (Santa Cruz Biotechnology Inc.); or GAPDH (AbCam).

    Techniques: Expressing, Activity Assay, In Vitro, Protein Kinase A Assay, Western Blot

    An IP antagonist opposes iloprost-enhanced oxytocin-induced contractions and iloprost-induced contractile protein expression. ( A ) Human myometrial tissue in organ culture was pretreated with 90 μmol/l RO3244794 (RO) for 30 min and then stimulated with or without 25 nmol/l iloprost for 48 h, after which tissue was transferred to an isometric muscle bath and treated with 5 nmol/l oxytocin as described in Methods. Average peak contraction data are represented as fold change relative to vehicle control. Arithmetic mean values are represented by bars, and individual measurements are plotted for the 3 patients. ( B ) Representative tracings for 1 patient in A . ( C ) Homogenates of uterine tissue isolated from A were subjected to Western blot analysis with antibodies to h-caldesmon, connexin 43, GAPDH, or β-tubulin. ( D ) Human myometrial tissue in organ culture was pretreated with 90 μmol/l RO3244794 for 30 min and then treated with vehicle or 25 nmol/l iloprost for 48 h, followed by treatment with 5 nmol/l oxytocin for 8 h, as indicated. Protein was isolated from the tissue and subjected to Western blot analysis with antibodies to SM2-MHC or GAPDH. Lanes were run on the same gel but were noncontiguous (lines).

    Journal: The Journal of Clinical Investigation

    Article Title: Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

    doi: 10.1172/JCI33800

    Figure Lengend Snippet: An IP antagonist opposes iloprost-enhanced oxytocin-induced contractions and iloprost-induced contractile protein expression. ( A ) Human myometrial tissue in organ culture was pretreated with 90 μmol/l RO3244794 (RO) for 30 min and then stimulated with or without 25 nmol/l iloprost for 48 h, after which tissue was transferred to an isometric muscle bath and treated with 5 nmol/l oxytocin as described in Methods. Average peak contraction data are represented as fold change relative to vehicle control. Arithmetic mean values are represented by bars, and individual measurements are plotted for the 3 patients. ( B ) Representative tracings for 1 patient in A . ( C ) Homogenates of uterine tissue isolated from A were subjected to Western blot analysis with antibodies to h-caldesmon, connexin 43, GAPDH, or β-tubulin. ( D ) Human myometrial tissue in organ culture was pretreated with 90 μmol/l RO3244794 for 30 min and then treated with vehicle or 25 nmol/l iloprost for 48 h, followed by treatment with 5 nmol/l oxytocin for 8 h, as indicated. Protein was isolated from the tissue and subjected to Western blot analysis with antibodies to SM2-MHC or GAPDH. Lanes were run on the same gel but were noncontiguous (lines).

    Article Snippet: Equal amounts of protein (10–20 μg) from each lysate were subjected to Western blot analysis as described previously ( ) using primary antibodies against α-SMA, calponin, or h-caldesmon (Sigma-Aldrich); SM2-MHC (Seikagaku America); connexin 43 (Cell Signaling); PKA Cα, PKA Cβ, or β-tubulin (Santa Cruz Biotechnology Inc.); or GAPDH (AbCam).

    Techniques: Expressing, Organ Culture, Isolation, Western Blot

    Iloprost induces expression of contractile apparatus proteins in human uSMCs. ( A ) Cell lysates from human uSMCs treated with the indicated concentrations of iloprost for 8 h were subjected to Western blot analysis with antibodies to α-SMA, calponin, h-caldesmon, and SM2-MHC, or β-tubulin as a loading control. ( B – D ) Human uSMCs were treated with vehicle or 2.5 nmol/l iloprost for the indicated times. Cell lysates were subjected to Western blot analysis with antibodies to ( B ) calponin, ( C ) h-caldesmon, and ( D ) SM2-MHC; shown is 1 representative immunoblot for each antibody and quantified protein expression, corrected to β-tubulin and presented as fold induction in order to average data from multiple experiments. n is indicated for each time point. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

    doi: 10.1172/JCI33800

    Figure Lengend Snippet: Iloprost induces expression of contractile apparatus proteins in human uSMCs. ( A ) Cell lysates from human uSMCs treated with the indicated concentrations of iloprost for 8 h were subjected to Western blot analysis with antibodies to α-SMA, calponin, h-caldesmon, and SM2-MHC, or β-tubulin as a loading control. ( B – D ) Human uSMCs were treated with vehicle or 2.5 nmol/l iloprost for the indicated times. Cell lysates were subjected to Western blot analysis with antibodies to ( B ) calponin, ( C ) h-caldesmon, and ( D ) SM2-MHC; shown is 1 representative immunoblot for each antibody and quantified protein expression, corrected to β-tubulin and presented as fold induction in order to average data from multiple experiments. n is indicated for each time point. * P

    Article Snippet: Equal amounts of protein (10–20 μg) from each lysate were subjected to Western blot analysis as described previously ( ) using primary antibodies against α-SMA, calponin, or h-caldesmon (Sigma-Aldrich); SM2-MHC (Seikagaku America); connexin 43 (Cell Signaling); PKA Cα, PKA Cβ, or β-tubulin (Santa Cruz Biotechnology Inc.); or GAPDH (AbCam).

    Techniques: Expressing, Western Blot

    Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Reduced centrosomal γ-tubulin staining is specifically induced by VPS4 at the centrosome and is unaffected by ESCRT III depletion. ( a ) NIH3T3 cells were transfected with one of the indicated plasmids or siRNA construct. Fixed cells were immunostained for γ-tubulin (blue) and imaged using 3D SIM. Maximum intensity projections of representative cells are shown. GFP n = 20, scRNA n = 17, GFP- VPS4 EQ n = 21, siVPS4A/B n = 13, VPS4 EQΔMIT n = 11, siCHMP2A n = 20, siCHMP2B n = 12 and siCHMP4B n = 20. Data for each condition was obtained from at least two independent experiments. Scale, 0.2 μm. ( b ) Cells were transfected with GFP-VPS4 EQ alone (upper panel) or together with ESCRT-III components (middle and bottom panels). Fixed cells were immunostained for γ-tubulin and imaged using 3D SIM. Maximum intensity projections of reconstructed images from representative cells are shown. Left to right: an overlay image of the entire cell (scale, 10 μm), zoomed in images of the centrosome (white box): ESCRT-III (red), VPS4 EQ (green) or γ-tubulin (blue) and an overlay (scale, 0.5 μm). GFP-VPS4 EQ n = 21, co-transfection with mCherry-CHMP2A n = 12, co-transfection with mCherry-CHMP4B n = 9. ( c ) 3D volume of centrosomal γ-tubulin structure was calculated in each cell using Volocity image analysis package. Statistical analysis for average volume was calculated using a one-way ANOVA. ***p- value ≤ 0.0001. ( d ). ( e , f ) NIH3T3 cells transfected with GFP or GFP-VPS4 EQ were either harvested 24 h post transfection and subjected to western blot analysis using anti-NEDD1 antibodies ( e ), or fixed and immunostained with anti-NEDD1 antibodies ( f ). Top to bottom in ( f ): an overlay image of the entire cell (scale, 5 μm), zoomed in images of the centrosome (white box): NEDD1 (red) and an overlay (scale, 0.2 μm) GFP n = 46, GFP-VPS4 EQ n = 34. ( g ) 3D volume of centrosomal NEDD1 in each cell was calculated using Volocity image analysis package. Statistical analysis for average volume was calculated using t-test. ***p- value ≤ 0.0001.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Staining, Transfection, Construct, Cotransfection, Western Blot

    ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: ESCRT-III components do not recruit VPS4 to centrosomes. ( a ) Maximum projection images of representative NIH3T3 cells, transfected with Pericentrin–RFP, and the indicated plasmids or immunostained for the endogenous ESCRT-III proteins CHMP2A or CHMP2B are shown. Top to bottom: entire cell (scale 10 μm), zoomed-in images of the centrosome (white box): ESCRT-III component (green), Pericentrin (red), an overlay (scale, 1 μm) and a line intensity profile of both channels along the centrosome. ( b ). n > 100, from at least two independent experiments. ( c ) NIH3T3 cells expressing GFP-VPS4 EQ were fixed, immunostained with the indicated ESCRT-III antibodies and imaged using a confocal spinning disk microscope. Shown are representative images (data obtained from at least two independent experiments for each protein tested). Top to bottom: entire cell (scale, 10 μm), zoomed-in images (white box) of: ESCRT-III (red), VPS4 EQ (green) and an overlay (scale, 1 μm). ( d ) NIH3T3 cells, transfected with GFP-VPS4 EQ and the indicated ESCRT III components, were immunostained with anti γ-tubulin and imaged. Shown are images of representative cells. Upper panel: entire cells (scale, 10 μm). Zoomed-in images (marked by squares in upper panel) of MVBs (left; yellow) and the centrosome (right; white) are shown below: VPS4 EQ (green), ESCRT III (red), γ-tubulin (blue) (scale 1 μm). Co-transfection with mCherry-CHMP2A n = 12, co- transfection with mCherry-CHMP4B n = 9. ( e ) VPS4 is recruited to the centrosome independent of ESCRT III. NIH3T3 cells were co-transfected with GFP-VPS4 EQ and mCherry-CHMP6-N peptide (composed of the first 52 amino acids of CHMP6) (upper panel) or with siCHMP4B and GFP-VPS4 EQ (bottom panel). Cells were then fixed, immunostained with γ-tubulin antibodies and imaged. Left to right: entire cell (scale, 10 μm), zoomed-in images (white box) of: γ-tubulin (blue), VPS4 EQ (green) and an overlay (scale, 1 μm). White arrows mark the centrosome. CHMP6-N n = 21, siCHMP4B n = 13.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Transfection, Expressing, Microscopy, Cotransfection

    Ciliogenesis is defective in cells with altered VPS4 activity. ( a ) NIH3T3 transfected with the indicated plasmids or siRNA constructs were fixed, immunostained with anti-acetylated antibodies and imaged. The percentage of ciliated cells is displayed in the graph (short cilia ≤ 2 µm, cilia ≥ 2 µm). Statistical analysis was calculated using a one-way ANOVA (***p- value ≤ 0.0001). Maximum intensity projection images of representative cells are shown in the right panel. Arrows indicate cilia (scale, 10 μm). scRNA n = 113, siVPS4A/B n = 103, siCHMP2A n = 64, siCHMP2B n = 173, siCHMP4B n = 68, GFP-VPS4 EQΔMIT n = 185. Data was obtained from at least two independent experiments. GFP and GFP-VPS4 EQ transfections were repeated in each experiment for reference. For these conditions data was obtained from more than 10 experiments, n ≥ 700. ( b ) Zebrafish embryos were injected with mRNA encoding either GFP or GFP-VPS4 EQ . 32 h post injection embryos were analyzed for survival (GFP n = 112, GFP-VPS4 EQ n = 132). Live embryos were analyzed for developmental defects (GFP n = 71, GFP-VPS4 EQ n = 21). Representative images are shown. Scale, 100 μm. ( c ) Embryos (15 h) were fixed and immunostained with acetylated-tubulin antibodies. The number of cilia in the Kupffer’s vesicle of each animal was counted and embryos were categorized as control (not injected or injected with GFP), GFP-VPS4 EQ injected developmentally normal, or GFP-VPS4 EQ injected with developmental defects. Control n = 41, GFP-VPS4 EQ normal n = 17, GFP-VPS4 EQ defective n = 31. Statistical analysis was calculated using t-test. *p- value ≤ 0.1, **p- value ≤ 0.05. Scale, 10 μm. ( d ). Cells were then treated and serially sectioned as described in material and methods and imaged by TEM. Left panel: representative image of a GFP expressing cells. Second to fourth panels: representative images of cells expressing GFP-VPS4 EQ . GFP n = 16, GFP-VPS4 EQ n = 19. Scale, 0.2 μm. Right panel: percentage of ciliated cells and cells with a docked ciliary vesicle. ( e ) NIH3T3 cells, expressing PACT-TagBFP (blue) and GFP-VPS4 EQ (green) and immunostained for the cilia marker Arl13b (red) were imaged using Airyscan confocal microscopy. Scale, 0.5 μm. ( f ) NIH cells transfected with mCherry-VPS4 EQ (green) and PACT tagBFP (blue) and Smo GFP (green) were imaged live using Airyscan microscopy. Scale, 0.5 μm.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Ciliogenesis is defective in cells with altered VPS4 activity. ( a ) NIH3T3 transfected with the indicated plasmids or siRNA constructs were fixed, immunostained with anti-acetylated antibodies and imaged. The percentage of ciliated cells is displayed in the graph (short cilia ≤ 2 µm, cilia ≥ 2 µm). Statistical analysis was calculated using a one-way ANOVA (***p- value ≤ 0.0001). Maximum intensity projection images of representative cells are shown in the right panel. Arrows indicate cilia (scale, 10 μm). scRNA n = 113, siVPS4A/B n = 103, siCHMP2A n = 64, siCHMP2B n = 173, siCHMP4B n = 68, GFP-VPS4 EQΔMIT n = 185. Data was obtained from at least two independent experiments. GFP and GFP-VPS4 EQ transfections were repeated in each experiment for reference. For these conditions data was obtained from more than 10 experiments, n ≥ 700. ( b ) Zebrafish embryos were injected with mRNA encoding either GFP or GFP-VPS4 EQ . 32 h post injection embryos were analyzed for survival (GFP n = 112, GFP-VPS4 EQ n = 132). Live embryos were analyzed for developmental defects (GFP n = 71, GFP-VPS4 EQ n = 21). Representative images are shown. Scale, 100 μm. ( c ) Embryos (15 h) were fixed and immunostained with acetylated-tubulin antibodies. The number of cilia in the Kupffer’s vesicle of each animal was counted and embryos were categorized as control (not injected or injected with GFP), GFP-VPS4 EQ injected developmentally normal, or GFP-VPS4 EQ injected with developmental defects. Control n = 41, GFP-VPS4 EQ normal n = 17, GFP-VPS4 EQ defective n = 31. Statistical analysis was calculated using t-test. *p- value ≤ 0.1, **p- value ≤ 0.05. Scale, 10 μm. ( d ). Cells were then treated and serially sectioned as described in material and methods and imaged by TEM. Left panel: representative image of a GFP expressing cells. Second to fourth panels: representative images of cells expressing GFP-VPS4 EQ . GFP n = 16, GFP-VPS4 EQ n = 19. Scale, 0.2 μm. Right panel: percentage of ciliated cells and cells with a docked ciliary vesicle. ( e ) NIH3T3 cells, expressing PACT-TagBFP (blue) and GFP-VPS4 EQ (green) and immunostained for the cilia marker Arl13b (red) were imaged using Airyscan confocal microscopy. Scale, 0.5 μm. ( f ) NIH cells transfected with mCherry-VPS4 EQ (green) and PACT tagBFP (blue) and Smo GFP (green) were imaged live using Airyscan microscopy. Scale, 0.5 μm.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Activity Assay, Transfection, Construct, Injection, Transmission Electron Microscopy, Expressing, Marker, Confocal Microscopy, Microscopy

    A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: A model for VPS4 function at centrosomes. ( a ) In normal conditions VPS4 dynamically associates with the centrosome. This dynamic VPS4 localization ensures proper γ-tubulin organization and MT growth at the centrosome where it also facilitates ciliogenesis. ( b ) Inhibition of VPS4 dynamic association with the centrosome using the ATP locked mutant VPS4 EQ , leads to reduced γ-tubulin levels and loss of γ-tubulin ring structure at the centrosome. Consequently, MT growth from centrosomes is impaired, centrosome positioning is misregulated, centriolar satellites are lost, and ciliogenesis is inhibited.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Inhibition, Mutagenesis

    Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Expression of VPS4 EQ causes reduced γ-tubulin staining at centrosomes but does not affect overall centriolar structure. ( a ) NIH3T3 cells grown on gridded coverslips were fixed and imaged to locate cells expressing GFP or GFP-VPS4 EQ ). Scale, 0.2 μm. ( b–d ) The organization of known centrosomal proteins was tested in fixed NIH3T3 cells expressing GFP (control), GFP-VPS4 or GFP- VPS4 EQ , immunostained with the indicated antibodies. Cells were imaged using 3D SIM. Shown are maximum intensity projections of reconstructed images from representative cells. Each panel shows (from left to right) the entire cell (scale, 5 μm); zoomed-in images (white box) of each channel and a zoomed-in overlay image (scale, 0.2 μm). ( b ) Endogenous CP110 (antibody staining, blue) GFP n = 59, VPS4 n = 10, VPS4 EQ n = 20. ( c ) Endogenous Cep164 (antibody staining, blue) GFP n = 10, GFP-VPS4 n = 8, GFP- VPS4 EQ n = 15. ( d ) Endogenous γ-tubulin (antibody staining, blue). GFP n = 20, GFP-VPS4 n = 15, GFP-VPS4 EQ n = 21. Note that while CP110 and Cep164 are not affected by VPS4 EQ expression, γ tubulin staining is severely reduced.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Expressing, Staining

    Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Radial MT growth and centrosome positioning are abnormal upon perturbation of VPS4 ATPase activity. ( a ) NIH3T3 cells were co-transfected with EB1-GFP, a MT plus-end binding protein, and with either mCherry or mCherry-VPS4 EQ ). mCherry n = 14, mCherry-VPS4 EQ n = 19. Graph on right: percentage of cells in which radial MT growth was observed. Statistical analysis for normal radial MT was calculated using t-test ***p- value ≤ 0.0001. Scale, 10 μm. ( b ) Fixed NIH3T3 cells expressing either GFP or GFP-VPS4 EQ were immunostained with anti-acetylated tubulin (red) and anti γ-tubulin (blue) antibodies and imaged in 3D using SIM. Maximum intensity projections of representative images are shown. GFP n = 147, GFP-VPS4 EQ n = 115. White arrows indicate the centrosome. Graph on right: percentage of cells exhibiting normal or heavy acetylation. Statistical analysis for normal acetylation was calculated using t-test ***p- value ≤ 0.0001. Scale, 1 μm. ( c ) NIH3T3 cells were transfected with the centrosome marker PACT-mRFP (red) and with either GFP or GFP-VPS4 EQ (green). 24 h post transfection cells were plated on fibronectin coated micropatterns (as described in materials and methods), fixed, stained with Hoechst (blue) and imaged in 3D using a spinning disk confocal microscope. Shown are maximum intensity Y projection images of representative cells. The distance between the centrosome and the center of the nucleus (see cartoon on the right) was measured in 3D for each cell as described in materials and methods and plotted in a histogram (bottom panel). GFP n = 45, GFP-VPS4 EQ n = 50. Scale, 10 μm.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Activity Assay, Transfection, Binding Assay, Expressing, Marker, Staining, Microscopy

    Localization and dynamics of VPS4 at the centrosomes of interphase cells. ( a ) NIH3T3 cells were transfected with the centrosome marker, PACT-mRFP, and GFP-VPS4 or GFP-VPS4 EQ and imaged by Airyscan confocal microscopy. Images were processed to reveal the sub-diffraction localization of the proteins. Both the wild-type and the ATPase deficient VPS4 localize to the centrosome. Left to right: entire cell (scale, 5 μm), zoomed-in images (white box) of: PACT (red), VPS4 (green), an overlay and a YZ projection (scale, 0.5 μm). ( b ) Top panel: NIH3T3 cells transfected with GFP, GFP-VPS4 or GFP-VPS4 EQ ). GFP-VPS4 and GFP-VPS4 EQ express at similar levels. Bottom panel: Cells expressing GFP, GFP-VPS4, or GFP-VPS4 EQ together with PACT-TagBFP were fixed and stained with anti-centrin. Z stacks of centrosomes were collected based on centrosomal markers using Airyscan microscopy. GFP intensity at the centrosome was quantified as described in materials and methods. Shown are average intensity values that were normalized to average GFP intensity obtained in control cells. GFP n = 56, GFP-VPS4 n = 35, GFP-VPS4 EQ n = 52 from 2 independent experiments. ( c ) Cells transfected with PACT-mRFP and GFP-VPS4 or GFP-VPS4 EQ were grown in high glucose media, fixed, and stained with Hoecst and antibodies to acetylated tubulin. GFP-VPS4 EQ expressing cells in anaphase or metaphase like the one shown here were rare. Scale, whole cells, 5 μm; zoomed-in image, 1 μm. ( d ) iFRAP recordings of NIH3T3 cells expressing PACT-mRFP and either GFP-VPS4 or GFP-VPS4 EQ were performed using Airyscan Confocal imaging in Fast mode. A single plane was imaged and then two regions on either side of the centrosome (covering most of the cell) were photobleached (white frame box). Image capture alternated with photobleaching for 90–100 time points. The intensity of the GFP signal at the centrosome was quantified using the location of the PACT-mRFP marker to generate the analysis region (small yellow frame box). Intensity was normalized to the pre-bleach intensity and the mean+/− the standard deviation was plotted for GFP-VPS4 and GFP-VPS4 EQ (top right panel). Bottom panel: zoomed-in images taken from yellow frame boxes in the corresponding upper panels are shown. GFP-VPS4 n = 11, GFP-VPS4 EQ n = 5. Scale, upper panel, 5 μm; bottom panel, 0.5 μm.

    Journal: Scientific Reports

    Article Title: VPS4 is a dynamic component of the centrosome that regulates centrosome localization of γ-tubulin, centriolar satellite stability and ciliogenesis

    doi: 10.1038/s41598-018-21491-x

    Figure Lengend Snippet: Localization and dynamics of VPS4 at the centrosomes of interphase cells. ( a ) NIH3T3 cells were transfected with the centrosome marker, PACT-mRFP, and GFP-VPS4 or GFP-VPS4 EQ and imaged by Airyscan confocal microscopy. Images were processed to reveal the sub-diffraction localization of the proteins. Both the wild-type and the ATPase deficient VPS4 localize to the centrosome. Left to right: entire cell (scale, 5 μm), zoomed-in images (white box) of: PACT (red), VPS4 (green), an overlay and a YZ projection (scale, 0.5 μm). ( b ) Top panel: NIH3T3 cells transfected with GFP, GFP-VPS4 or GFP-VPS4 EQ ). GFP-VPS4 and GFP-VPS4 EQ express at similar levels. Bottom panel: Cells expressing GFP, GFP-VPS4, or GFP-VPS4 EQ together with PACT-TagBFP were fixed and stained with anti-centrin. Z stacks of centrosomes were collected based on centrosomal markers using Airyscan microscopy. GFP intensity at the centrosome was quantified as described in materials and methods. Shown are average intensity values that were normalized to average GFP intensity obtained in control cells. GFP n = 56, GFP-VPS4 n = 35, GFP-VPS4 EQ n = 52 from 2 independent experiments. ( c ) Cells transfected with PACT-mRFP and GFP-VPS4 or GFP-VPS4 EQ were grown in high glucose media, fixed, and stained with Hoecst and antibodies to acetylated tubulin. GFP-VPS4 EQ expressing cells in anaphase or metaphase like the one shown here were rare. Scale, whole cells, 5 μm; zoomed-in image, 1 μm. ( d ) iFRAP recordings of NIH3T3 cells expressing PACT-mRFP and either GFP-VPS4 or GFP-VPS4 EQ were performed using Airyscan Confocal imaging in Fast mode. A single plane was imaged and then two regions on either side of the centrosome (covering most of the cell) were photobleached (white frame box). Image capture alternated with photobleaching for 90–100 time points. The intensity of the GFP signal at the centrosome was quantified using the location of the PACT-mRFP marker to generate the analysis region (small yellow frame box). Intensity was normalized to the pre-bleach intensity and the mean+/− the standard deviation was plotted for GFP-VPS4 and GFP-VPS4 EQ (top right panel). Bottom panel: zoomed-in images taken from yellow frame boxes in the corresponding upper panels are shown. GFP-VPS4 n = 11, GFP-VPS4 EQ n = 5. Scale, upper panel, 5 μm; bottom panel, 0.5 μm.

    Article Snippet: Cells were then permeabilized, blocked and immunostained with the following primary antibodies as specified in text: mouse monoclonal anti α-tubulin antibodies (1:1000, DM1A clone Sigma-Aldrich Cat # T9026), mouse monoclonal anti acetylated tubulin antibodies (1:500 or 1:1000, Thermo Scientific Cat # 32–2700 or 1:1000, Sigma-Aldrich Cat # T7451), mouse monoclonal anti γ-tubulin (1:1000, Sigma-Aldrich Cat #T6557), mouse monoclonal anti NEDD1 (1:100, Abcam Cat # ab57336), rabbit polyclonal anti γ-tubulin antibodies (1:200, Abcam Cat # ab 84355), rabbit polyclonal anti CHMP2A (1:50, Proteintech Group Cat # 10477-1-AP) rabbit polyclonal anti CHMP2B (1:50, Proteintech Group Cat # 12527-1-AP), rabbit polyclonal anti IST1 (1:50, Proteintech Group Cat # 51002-1-AP), rabbit polyclonal anti CP110 (1:50, Proteintech Group Cat # 12780-1-AP) antibodies, rabbit polyclonal anti CHMP4A (1:50, Santa Cruz Biotechnology Cat # SC-67229), rabbit polyclonal anti PCM1 antibodies (1:100, Santa Cruz Biotechnology, Cat # SC-67204), rabbit polyclonal anti Cep164 (1: 200, Abcam Cat # ab106372 or 1:200 Proteintech Group Cat # 22227-1-AP), rabbit polyclonal anti Cep290 (1:100, Abcam Cat # ab84870), mouse anti Centrin (1:500, Millipore clone 20H5, Cat # 04-1624) and rabbit polyclonal anti VTA1 antibodies (1:50, Pierce Biotechnology Cat # PA521831).

    Techniques: Transfection, Marker, Confocal Microscopy, Expressing, Staining, Microscopy, Imaging, Standard Deviation