α-tubulin Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher α tubulin
    α Tubulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1509 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α tubulin/product/Thermo Fisher
    Average 99 stars, based on 1509 article reviews
    Price from $9.99 to $1999.99
    α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    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
    https://www.bioz.com/result/α tubulin/product/Millipore
    Average 99 stars, based on 21797 article reviews
    Price from $9.99 to $1999.99
    α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti alpha tubulin antibody
    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 ).
    Monoclonal Anti Alpha Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19941 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti alpha tubulin antibody/product/Millipore
    Average 99 stars, based on 19941 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti alpha tubulin antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Millipore anti α tubulin
    Fluorescence microscopy indicates defective cytokinesis in DU145 cells treated with plagiochiline A. DU145 cells were plated on glass cover slips and incubated 24 h at 37 °C. Cells were then treated for 48 h with 5 µM plagiochiline A or vehicle control (DMSO). Cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100. Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, blue) and <t>α-tubulin</t> was stained using anti-α-tubulin antibody labeled with fluorescein isothiocyanate (FITC, green). ( A ) Representative photomicrographs with arrows indicating cells arrested at late cytokinesis (i.e., nascent daughters remain attached by intercellular bridges). ( B ) Graph showing the number of mitotic figures observed per field examined (500 cells). Columns represent the mean of four independent experiments with bars representing standard error. Comparing plagiochiline A-treated vs. vehicle control cells, the increase in late cytokinesis and the decrease in other mitotic figures were statistically significant (*, P = 0.001 and 0.0084, respectively).
    Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11610 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti α tubulin/product/Millipore
    Average 99 stars, based on 11610 article reviews
    Price from $9.99 to $1999.99
    anti α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    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
    https://www.bioz.com/result/α tubulin/product/Santa Cruz Biotechnology
    Average 94 stars, based on 7178 article reviews
    Price from $9.99 to $1999.99
    α tubulin - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    99
    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
    https://www.bioz.com/result/α tubulin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 5143 article reviews
    Price from $9.99 to $1999.99
    α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    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
    https://www.bioz.com/result/mouse anti α tubulin/product/Millipore
    Average 99 stars, based on 5918 article reviews
    Price from $9.99 to $1999.99
    mouse anti α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    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
    https://www.bioz.com/result/α tubulin/product/Abcam
    Average 99 stars, based on 4261 article reviews
    Price from $9.99 to $1999.99
    α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc 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.
    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
    https://www.bioz.com/result/anti α tubulin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1538 article reviews
    Price from $9.99 to $1999.99
    anti α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Millipore α tubulin ab
    Lentiviral-mediated overexpression of α-syn. ( A ) Western blot analysis shows the overexpression of normal and mutated (A53T and A30P) human and rat α-syn in the SH-SY5Y human neuroblastoma cell line. All α-syn forms are expressed at similar levels for the same amount of viral particles. Protein (25 μg per lane) were loaded for the noninfected cells (NI) and cells transduced with lentiviral vectors encoding for cytoplasmic LacZ, rat α-syn, wild-type (HWT), and mutated forms of human α-syn. The 19-kDa α-syn bands (α-syn) were detected with a polyclonal rabbit Ab generated against the 101- to 124-aa sequence of human α-syn. This Ab recognizes both human and rat α-syn on Western blot. The amount of protein loaded was checked by reprobing the same membrane with an <t>α-tubulin</t> Ab (α-tub). ( B – D ) Lentiviral vectors encoding for wild-type and mutated human α-syn were stereotactically injected in the substantia nigra of rats. The nigral dopaminergic neurons were specifically labeled with a TH Ab ( B ). Detection with an α-syn polyclonal Ab revealed a significant overexpression of A30P α-syn ( C ) in the injected hemisphere. No α-syn staining was observed on the contralateral side. Double staining ( D , yellow-orange color) shows a large proportion of TH-IR neurons overexpressing α-syn. (Scale bars = 200 μm.)
    α Tubulin Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α tubulin ab/product/Millipore
    Average 99 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    α tubulin ab - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Abcam anti α tubulin
    Induction of morphological change and suppression of migration by low eribulin concentrations ( A ) Immunofluorescent images of LM8 cells stained for <t>α-tubulin</t> (green) and nucleus (blue). LM8 cells were treated with eribulin for 16 h. LM8 cells became round and lost their cell protrusions. Scale bar: 10 μ m. ( B ) Phase-contrast images showing dose-dependent changes in the morphology of LM8 cells treated with eribulin (left). Number of protrusions on LM8 cells (right). Values are mean ± SEMs (≥30 cells per group). ** P
    Anti α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti α tubulin/product/Abcam
    Average 99 stars, based on 1354 article reviews
    Price from $9.99 to $1999.99
    anti α tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    96
    Cell Signaling Technology Inc anti tubulin
    CG30463/pgant9 encodes a Golgi-localized O-glycosyltransferase. a Gene structure for CG30463/pgant9 is shown, with boxes representing exons and lines representing introns. The N-terminal (blue), catalytic (orange) and lectin (green) domains of the putative glycosyltransferase encoded by CG30463 are shown. The lectin domain consists of three subdomains (α, β, and γ). The sequence for the differentially spliced α subdomain (exon 8) is shown, with acidic residues highlighted in red and basic residues highlighted in blue. b Both splice variants (V5-tagged; red) localized to the Golgi apparatus (as detected by anti-GM130; blue) in S2R+ cells. Scale bar, 10 μm. Representative images from two independent experiments are shown. c Western blots of S2R+ cells expressing vector alone (Vector), a V5-tagged recombinant CG30463A or a V5-tagged recombinant CG30463B . Panels on the left show CG30463A and CG30463B expression with the V5-tag (anti-V5) and loading controls <t>(anti-tubulin).</t> Panel on the right shows increased O-glycosylation (as detected by the lectin HPA) when CG30463A or CG30463B are expressed in S2R+ cells. Representative western blots from three independent experiments are shown. Molecular weight markers (kD) are shown to the left of each panel
    Anti Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti tubulin/product/Cell Signaling Technology Inc
    Average 96 stars, based on 955 article reviews
    Price from $9.99 to $1999.99
    anti tubulin - by Bioz Stars, 2020-10
    96/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti tubulin acetylated antibody
    CG30463/pgant9 encodes a Golgi-localized O-glycosyltransferase. a Gene structure for CG30463/pgant9 is shown, with boxes representing exons and lines representing introns. The N-terminal (blue), catalytic (orange) and lectin (green) domains of the putative glycosyltransferase encoded by CG30463 are shown. The lectin domain consists of three subdomains (α, β, and γ). The sequence for the differentially spliced α subdomain (exon 8) is shown, with acidic residues highlighted in red and basic residues highlighted in blue. b Both splice variants (V5-tagged; red) localized to the Golgi apparatus (as detected by anti-GM130; blue) in S2R+ cells. Scale bar, 10 μm. Representative images from two independent experiments are shown. c Western blots of S2R+ cells expressing vector alone (Vector), a V5-tagged recombinant CG30463A or a V5-tagged recombinant CG30463B . Panels on the left show CG30463A and CG30463B expression with the V5-tag (anti-V5) and loading controls <t>(anti-tubulin).</t> Panel on the right shows increased O-glycosylation (as detected by the lectin HPA) when CG30463A or CG30463B are expressed in S2R+ cells. Representative western blots from three independent experiments are shown. Molecular weight markers (kD) are shown to the left of each panel
    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
    https://www.bioz.com/result/monoclonal anti tubulin acetylated antibody/product/Millipore
    Average 99 stars, based on 3808 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti tubulin acetylated antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc tubulin
    CG30463/pgant9 encodes a Golgi-localized O-glycosyltransferase. a Gene structure for CG30463/pgant9 is shown, with boxes representing exons and lines representing introns. The N-terminal (blue), catalytic (orange) and lectin (green) domains of the putative glycosyltransferase encoded by CG30463 are shown. The lectin domain consists of three subdomains (α, β, and γ). The sequence for the differentially spliced α subdomain (exon 8) is shown, with acidic residues highlighted in red and basic residues highlighted in blue. b Both splice variants (V5-tagged; red) localized to the Golgi apparatus (as detected by anti-GM130; blue) in S2R+ cells. Scale bar, 10 μm. Representative images from two independent experiments are shown. c Western blots of S2R+ cells expressing vector alone (Vector), a V5-tagged recombinant CG30463A or a V5-tagged recombinant CG30463B . Panels on the left show CG30463A and CG30463B expression with the V5-tag (anti-V5) and loading controls <t>(anti-tubulin).</t> Panel on the right shows increased O-glycosylation (as detected by the lectin HPA) when CG30463A or CG30463B are expressed in S2R+ cells. Representative western blots from three independent experiments are shown. Molecular weight markers (kD) are shown to the left of each panel
    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
    https://www.bioz.com/result/tubulin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2637 article reviews
    Price from $9.99 to $1999.99
    tubulin - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Millipore monoclonal anti acetylated tubulin antibody
    Mutations in MNS1 when combined with mutations in DNAH5 might result in defects of the ODA-microtubule docking complex in human respiratory epithelial cells. (A) Transmission electron micrographs show subtle ultrastructural defects in affected individual AL-III-9 carrying bi-allelic MNS1 mutations with the occasional absence of only few ODAs (2–4 out of 9) in about half of the cross-sections (compared to control samples where all analyzed sections show an average of 8.7 ODAs, 9 analyzed sections from the MNS1-deficient ciliary axonemes show an average of 6 ODAs). However, TEM show complete absence of ODAs in PCD-affected individuals OI-24 II1 ( DNAH5 mutations) and OI-11 II6 ( MNS1 and DNAH5 mutations) compared to a control without PCD. In the healthy control, outer dynein arms are visible (blue arrows). However, the cilia from OI-24 II1 still have the ODA-DC (small projections marked by white arrows) whereas the cilia from OI-11 II6 do not, suggesting that MNS1 deficiency when combined with DNAH5 deficiency might cause defects in ODA-DC assembly. Below the control TEM section a schematic illustrating a microtubular doublet with attached ODA docking complex (ODA-DC) and the double-headed ODA complex proteins with dynein heavy chain DNAH5 and dynein intermediate chains DNAI1 and DNAI2. In affected individual AL-III-9, a partial defect is observed; in OI-24II1, a schematic where the ODA complex is absent while the ODA-DC is still retained; in OI-11II6, a schematic where both ODA and ODA docking complexes are absent. Scale bars, 0.1 μm. (B) Respiratory epithelial cells from control and affected individuals: AL-III-9 carrying bi-allelic MNS1 mutations, OI-11 II6 carrying bi-allelic MNS1 and DNAH5 mutations and OI-24 II1 carrying no mutations in MNS1 but identical bi-allelic DNAH5 mutations as OI-11 II6. For space issues, OI-24 II1 is described as DNAH5 mut/mut instead of DNAH5 c . 13432_13435delCACT/ c . 13432_13435delCACT . Cells were double-labeled with antibodies directed against acetylated <t>alpha-tubulin</t> (green) and CCDC114 (HPA042524, Atlas antibodies) (red). Nuclei were stained with Hoechst 33342 (blue). Both proteins co-localize (yellow) along the ciliary axonemes in cells from the unaffected controls, AL-III-9 and OI-24 II1, while in cells of OI-11 II6, CCDC114 localizes only to the proximal part of the ciliary axonemes, indicating that recessive loss-of-function mutations in MNS1 when combined with loss-of-function mutations in DNAH5 might affect the distal localization of ODA-DC associated proteins and might play a role in docking or anchoring the ODA subunits or in regulating this process. Scale bars, 10μm.
    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
    https://www.bioz.com/result/monoclonal anti acetylated tubulin antibody/product/Millipore
    Average 99 stars, based on 2829 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti acetylated tubulin antibody - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    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

    Fluorescence microscopy indicates defective cytokinesis in DU145 cells treated with plagiochiline A. DU145 cells were plated on glass cover slips and incubated 24 h at 37 °C. Cells were then treated for 48 h with 5 µM plagiochiline A or vehicle control (DMSO). Cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100. Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, blue) and α-tubulin was stained using anti-α-tubulin antibody labeled with fluorescein isothiocyanate (FITC, green). ( A ) Representative photomicrographs with arrows indicating cells arrested at late cytokinesis (i.e., nascent daughters remain attached by intercellular bridges). ( B ) Graph showing the number of mitotic figures observed per field examined (500 cells). Columns represent the mean of four independent experiments with bars representing standard error. Comparing plagiochiline A-treated vs. vehicle control cells, the increase in late cytokinesis and the decrease in other mitotic figures were statistically significant (*, P = 0.001 and 0.0084, respectively).

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Plagiochiline A Inhibits Cytokinetic Abscission and Induces Cell Death

    doi: 10.3390/molecules23061418

    Figure Lengend Snippet: Fluorescence microscopy indicates defective cytokinesis in DU145 cells treated with plagiochiline A. DU145 cells were plated on glass cover slips and incubated 24 h at 37 °C. Cells were then treated for 48 h with 5 µM plagiochiline A or vehicle control (DMSO). Cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100. Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, blue) and α-tubulin was stained using anti-α-tubulin antibody labeled with fluorescein isothiocyanate (FITC, green). ( A ) Representative photomicrographs with arrows indicating cells arrested at late cytokinesis (i.e., nascent daughters remain attached by intercellular bridges). ( B ) Graph showing the number of mitotic figures observed per field examined (500 cells). Columns represent the mean of four independent experiments with bars representing standard error. Comparing plagiochiline A-treated vs. vehicle control cells, the increase in late cytokinesis and the decrease in other mitotic figures were statistically significant (*, P = 0.001 and 0.0084, respectively).

    Article Snippet: The fixed cells were incubated for 60 min at 37 °C with anti-α-tubulin monoclonal antibody (mouse IgG1 isotype) conjugated to fluorescein isothiocyanate, isomer I (FITC) from Sigma (dilution 1:100).

    Techniques: Fluorescence, Microscopy, Incubation, Staining, Labeling

    Antisense locked nucleic acid (LNA) therapeutic approach in Unverricht-Lundborg disease (ULD), patient-derived fibroblasts. ( A ) Schematic representation of the splicing downregulation observed in the presence of the c.66G > A CSTB mutation. The sequence of the LNA complementary to the cryptic donor site activated by the mutation and designed in order to block the recognition of the intronic alternative 5′ss in fibroblasts from the patient is shown. ( B ) Transcriptional profile obtained for HC and patient fibroblasts untreated (0 nM) and treated with quantities between 5 to 100 nM of LNA oligonucleotide. The RT-PCR analysis showed the disappearance of the aberrantly spliced transcript (451 bp) when cells were treated with 100 nM of the LNA oligonucleotide. Correctly spliced mRNA was obtained 24 h after transfection in a dose-dependent manner. ( C ) CSTB protein expression in control and patient fibroblasts untreated (0 nM) and treated with 100 nM of the LNA. The α-tubulin protein was used as loading control. M—molecular marker; NC—negative control; HC—healthy control.

    Journal: Genes

    Article Title: Correction of a Splicing Mutation Affecting an Unverricht-Lundborg Disease Patient by Antisense Therapy

    doi: 10.3390/genes9090455

    Figure Lengend Snippet: Antisense locked nucleic acid (LNA) therapeutic approach in Unverricht-Lundborg disease (ULD), patient-derived fibroblasts. ( A ) Schematic representation of the splicing downregulation observed in the presence of the c.66G > A CSTB mutation. The sequence of the LNA complementary to the cryptic donor site activated by the mutation and designed in order to block the recognition of the intronic alternative 5′ss in fibroblasts from the patient is shown. ( B ) Transcriptional profile obtained for HC and patient fibroblasts untreated (0 nM) and treated with quantities between 5 to 100 nM of LNA oligonucleotide. The RT-PCR analysis showed the disappearance of the aberrantly spliced transcript (451 bp) when cells were treated with 100 nM of the LNA oligonucleotide. Correctly spliced mRNA was obtained 24 h after transfection in a dose-dependent manner. ( C ) CSTB protein expression in control and patient fibroblasts untreated (0 nM) and treated with 100 nM of the LNA. The α-tubulin protein was used as loading control. M—molecular marker; NC—negative control; HC—healthy control.

    Article Snippet: The total amount of protein loaded was controlled by incubation with monoclonal anti-α-tubulin antibody (T6199—Sigma-Aldrich, St. Gallen, Switzerland).

    Techniques: Derivative Assay, Mutagenesis, Sequencing, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, Marker, Negative Control

    Visualisation of the excretory system of Paradiplozoon homoion adults using α-tubulin immunolabeling. A) Micrograph showing the distribution of flame cells and peripheral nerve fibres in the forebody region. CLSM, IFA-FITC. B) Flame cells (e ncircled ) counterstained with Hoechst to indicate the nuclei of terminal cells. Note the green stained ciliated tufts and rootlets. CLSM, IFA-FITC/Hoechst. C) Detail of the flame cells. The barrel non-ciliated part involves both the terminal and adjacent canal cell. CLSM, phalloidin-TRITC. D) Detail of one flame cell with the ciliated tuft of the terminal cell. CLSM, IFA-FITC. E) Detail of two flame cells. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. A and B are single median optical sections, while C and D are composite views created by flattening a series of optical sections. black arrowheads –flame cells, black arrows –roots of tuft cilia, white arrows –transverse connective cords.

    Journal: PLoS ONE

    Article Title: Architecture of Paradiplozoon homoion: A diplozoid monogenean exhibiting highly-developed equipment for ectoparasitism

    doi: 10.1371/journal.pone.0192285

    Figure Lengend Snippet: Visualisation of the excretory system of Paradiplozoon homoion adults using α-tubulin immunolabeling. A) Micrograph showing the distribution of flame cells and peripheral nerve fibres in the forebody region. CLSM, IFA-FITC. B) Flame cells (e ncircled ) counterstained with Hoechst to indicate the nuclei of terminal cells. Note the green stained ciliated tufts and rootlets. CLSM, IFA-FITC/Hoechst. C) Detail of the flame cells. The barrel non-ciliated part involves both the terminal and adjacent canal cell. CLSM, phalloidin-TRITC. D) Detail of one flame cell with the ciliated tuft of the terminal cell. CLSM, IFA-FITC. E) Detail of two flame cells. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. A and B are single median optical sections, while C and D are composite views created by flattening a series of optical sections. black arrowheads –flame cells, black arrows –roots of tuft cilia, white arrows –transverse connective cords.

    Article Snippet: The samples were then incubated with mouse monoclonal anti-α-tubulin antibody (Clone B-5-1-2, Sigma-Aldrich, Czech Republic) at 4°C for six days, washed for 24 h in AbD and finally incubated with mouse polyvalent immunoglobulins (1:125) in PBS with 1% BSA at 37°C for four days.

    Techniques: Immunolabeling, Confocal Laser Scanning Microscopy, Immunofluorescence, Staining

    Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on musculature. A) Lateral view of the haptor, with one row of four clamps and three lobed structures (one central and two lateral lobes). SEM. B) Muscular haptor equipped with four pairs of clamps with extrinsic muscle bundles and a central lobe. CLSM, phalloidin-TRITC. C) Central lobe of the haptor, with numerous flame cells. Note the muscle arrangement (longitudinal, circular and diagonal). CLSM, phalloidin-TRITC. D) Detail of the massive muscle bundles controlling the clamp. CLSM, phalloidin-TRITC. E) Detail of the clamp musculature surrounded by flame cells. Note the strong F-actin labelling localised in the barrel part of the flame cells. CLSM, phalloidin-TRITC. F) α-tubulin labelling of flame cell ciliated tufts located near the clamps. CLSM, IFA-FITC/DAPI. B - F represent composite views created by flattening a series of optical sections. black arrows –extrinsic muscle bundles, black arrowheads –flame cells, ce– central lobe, cl –clamps, cm –circular muscles, dm —diagonal muscles, la– lateral lobes, lm –longitudinal muscles, white arrowheads –sensory structures.

    Journal: PLoS ONE

    Article Title: Architecture of Paradiplozoon homoion: A diplozoid monogenean exhibiting highly-developed equipment for ectoparasitism

    doi: 10.1371/journal.pone.0192285

    Figure Lengend Snippet: Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on musculature. A) Lateral view of the haptor, with one row of four clamps and three lobed structures (one central and two lateral lobes). SEM. B) Muscular haptor equipped with four pairs of clamps with extrinsic muscle bundles and a central lobe. CLSM, phalloidin-TRITC. C) Central lobe of the haptor, with numerous flame cells. Note the muscle arrangement (longitudinal, circular and diagonal). CLSM, phalloidin-TRITC. D) Detail of the massive muscle bundles controlling the clamp. CLSM, phalloidin-TRITC. E) Detail of the clamp musculature surrounded by flame cells. Note the strong F-actin labelling localised in the barrel part of the flame cells. CLSM, phalloidin-TRITC. F) α-tubulin labelling of flame cell ciliated tufts located near the clamps. CLSM, IFA-FITC/DAPI. B - F represent composite views created by flattening a series of optical sections. black arrows –extrinsic muscle bundles, black arrowheads –flame cells, ce– central lobe, cl –clamps, cm –circular muscles, dm —diagonal muscles, la– lateral lobes, lm –longitudinal muscles, white arrowheads –sensory structures.

    Article Snippet: The samples were then incubated with mouse monoclonal anti-α-tubulin antibody (Clone B-5-1-2, Sigma-Aldrich, Czech Republic) at 4°C for six days, washed for 24 h in AbD and finally incubated with mouse polyvalent immunoglobulins (1:125) in PBS with 1% BSA at 37°C for four days.

    Techniques: Confocal Laser Scanning Microscopy, Immunofluorescence

    Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on innervation. A) Detail of the haptor central lobe. Note the distribution of uniciliated sensory structures (F-actin) and peripheral nerve fibre endings (α-tubulin). The micrograph shows the area marked by a white rectangle in C). CLSM, IFA-FITC/phalloidin-TRITC. B) Double F-actin and α-tubulin labelling of the region surrounding the two clamps. The micrograph shows the area marked by a red rectangle in C). The dense red structures represent autofluorescence of the clamp sclerites. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. C) General view of the hindbody labelled for α-tubulin and counterstained with Hoechst. CLSM, IFA-FITC/Hoechst. A - C represent composite views created by flattening a series of optical sections. black arrowheads –flame cells, black arrows –innervation of clamps, cl –clamps, white arrowheads –uniciliated sensory structures, white arrows –peripheral nerve fibres.

    Journal: PLoS ONE

    Article Title: Architecture of Paradiplozoon homoion: A diplozoid monogenean exhibiting highly-developed equipment for ectoparasitism

    doi: 10.1371/journal.pone.0192285

    Figure Lengend Snippet: Hindbody with haptor in Paradiplozoon homoion adults, with emphasis on innervation. A) Detail of the haptor central lobe. Note the distribution of uniciliated sensory structures (F-actin) and peripheral nerve fibre endings (α-tubulin). The micrograph shows the area marked by a white rectangle in C). CLSM, IFA-FITC/phalloidin-TRITC. B) Double F-actin and α-tubulin labelling of the region surrounding the two clamps. The micrograph shows the area marked by a red rectangle in C). The dense red structures represent autofluorescence of the clamp sclerites. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. C) General view of the hindbody labelled for α-tubulin and counterstained with Hoechst. CLSM, IFA-FITC/Hoechst. A - C represent composite views created by flattening a series of optical sections. black arrowheads –flame cells, black arrows –innervation of clamps, cl –clamps, white arrowheads –uniciliated sensory structures, white arrows –peripheral nerve fibres.

    Article Snippet: The samples were then incubated with mouse monoclonal anti-α-tubulin antibody (Clone B-5-1-2, Sigma-Aldrich, Czech Republic) at 4°C for six days, washed for 24 h in AbD and finally incubated with mouse polyvalent immunoglobulins (1:125) in PBS with 1% BSA at 37°C for four days.

    Techniques: Confocal Laser Scanning Microscopy, Immunofluorescence

    Mouth border of Paradiplozoon homoion adults. A) Median plane optical sectioning of the forebody. Note the accumulation of α-tubulin associated with the forebody apical part and buccal suckers. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. B) View of the forebody showing the tubulin-rich apical end. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. C) Detail of a uniciliated sensory structure with a raised circular rim and one long cilium. SEM. D-E) A different optical section of the specimen in B) revealing the tubulin-rich border of the mouth opening and the distribution of uniciliated sensory structures. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst (D) and IFA-FITC/Hoechst (E). F) Arrangement of the muscle fibres around the border of the mouth opening. CLSM, phalloidin-TRITC. G) Detail of uniciliated sensory structures in the forebody apical end. CLSM, IFA-FITC/DAPI. A-B, D-E and G are composite views created by flattening a series of optical sections, while F represents a single optical section. black arrow –raised circular rim, black arrowheads –flame cells, bs –buccal suckers, ph –pharynx, white arrow –cilium, white arrowheads –uniciliated sensory structure.

    Journal: PLoS ONE

    Article Title: Architecture of Paradiplozoon homoion: A diplozoid monogenean exhibiting highly-developed equipment for ectoparasitism

    doi: 10.1371/journal.pone.0192285

    Figure Lengend Snippet: Mouth border of Paradiplozoon homoion adults. A) Median plane optical sectioning of the forebody. Note the accumulation of α-tubulin associated with the forebody apical part and buccal suckers. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. B) View of the forebody showing the tubulin-rich apical end. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst. C) Detail of a uniciliated sensory structure with a raised circular rim and one long cilium. SEM. D-E) A different optical section of the specimen in B) revealing the tubulin-rich border of the mouth opening and the distribution of uniciliated sensory structures. CLSM, IFA-FITC/phalloidin-TRITC/Hoechst (D) and IFA-FITC/Hoechst (E). F) Arrangement of the muscle fibres around the border of the mouth opening. CLSM, phalloidin-TRITC. G) Detail of uniciliated sensory structures in the forebody apical end. CLSM, IFA-FITC/DAPI. A-B, D-E and G are composite views created by flattening a series of optical sections, while F represents a single optical section. black arrow –raised circular rim, black arrowheads –flame cells, bs –buccal suckers, ph –pharynx, white arrow –cilium, white arrowheads –uniciliated sensory structure.

    Article Snippet: The samples were then incubated with mouse monoclonal anti-α-tubulin antibody (Clone B-5-1-2, Sigma-Aldrich, Czech Republic) at 4°C for six days, washed for 24 h in AbD and finally incubated with mouse polyvalent immunoglobulins (1:125) in PBS with 1% BSA at 37°C for four days.

    Techniques: Confocal Laser Scanning Microscopy, Immunofluorescence

    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

    Lentiviral-mediated overexpression of α-syn. ( A ) Western blot analysis shows the overexpression of normal and mutated (A53T and A30P) human and rat α-syn in the SH-SY5Y human neuroblastoma cell line. All α-syn forms are expressed at similar levels for the same amount of viral particles. Protein (25 μg per lane) were loaded for the noninfected cells (NI) and cells transduced with lentiviral vectors encoding for cytoplasmic LacZ, rat α-syn, wild-type (HWT), and mutated forms of human α-syn. The 19-kDa α-syn bands (α-syn) were detected with a polyclonal rabbit Ab generated against the 101- to 124-aa sequence of human α-syn. This Ab recognizes both human and rat α-syn on Western blot. The amount of protein loaded was checked by reprobing the same membrane with an α-tubulin Ab (α-tub). ( B – D ) Lentiviral vectors encoding for wild-type and mutated human α-syn were stereotactically injected in the substantia nigra of rats. The nigral dopaminergic neurons were specifically labeled with a TH Ab ( B ). Detection with an α-syn polyclonal Ab revealed a significant overexpression of A30P α-syn ( C ) in the injected hemisphere. No α-syn staining was observed on the contralateral side. Double staining ( D , yellow-orange color) shows a large proportion of TH-IR neurons overexpressing α-syn. (Scale bars = 200 μm.)

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

    Article Title: ?-Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson's disease

    doi: 10.1073/pnas.152339799

    Figure Lengend Snippet: Lentiviral-mediated overexpression of α-syn. ( A ) Western blot analysis shows the overexpression of normal and mutated (A53T and A30P) human and rat α-syn in the SH-SY5Y human neuroblastoma cell line. All α-syn forms are expressed at similar levels for the same amount of viral particles. Protein (25 μg per lane) were loaded for the noninfected cells (NI) and cells transduced with lentiviral vectors encoding for cytoplasmic LacZ, rat α-syn, wild-type (HWT), and mutated forms of human α-syn. The 19-kDa α-syn bands (α-syn) were detected with a polyclonal rabbit Ab generated against the 101- to 124-aa sequence of human α-syn. This Ab recognizes both human and rat α-syn on Western blot. The amount of protein loaded was checked by reprobing the same membrane with an α-tubulin Ab (α-tub). ( B – D ) Lentiviral vectors encoding for wild-type and mutated human α-syn were stereotactically injected in the substantia nigra of rats. The nigral dopaminergic neurons were specifically labeled with a TH Ab ( B ). Detection with an α-syn polyclonal Ab revealed a significant overexpression of A30P α-syn ( C ) in the injected hemisphere. No α-syn staining was observed on the contralateral side. Double staining ( D , yellow-orange color) shows a large proportion of TH-IR neurons overexpressing α-syn. (Scale bars = 200 μm.)

    Article Snippet: The amount of protein loaded was checked by reprobing the same membrane with an α-tubulin Ab (1:2,000; Sigma).

    Techniques: Over Expression, Western Blot, Transduction, Generated, Sequencing, Injection, Labeling, Staining, Double Staining

    Induction of morphological change and suppression of migration by low eribulin concentrations ( A ) Immunofluorescent images of LM8 cells stained for α-tubulin (green) and nucleus (blue). LM8 cells were treated with eribulin for 16 h. LM8 cells became round and lost their cell protrusions. Scale bar: 10 μ m. ( B ) Phase-contrast images showing dose-dependent changes in the morphology of LM8 cells treated with eribulin (left). Number of protrusions on LM8 cells (right). Values are mean ± SEMs (≥30 cells per group). ** P

    Journal: Oncotarget

    Article Title: Low-dose eribulin reduces lung metastasis of osteosarcoma in vitro and in vivo

    doi: 10.18632/oncotarget.26536

    Figure Lengend Snippet: Induction of morphological change and suppression of migration by low eribulin concentrations ( A ) Immunofluorescent images of LM8 cells stained for α-tubulin (green) and nucleus (blue). LM8 cells were treated with eribulin for 16 h. LM8 cells became round and lost their cell protrusions. Scale bar: 10 μ m. ( B ) Phase-contrast images showing dose-dependent changes in the morphology of LM8 cells treated with eribulin (left). Number of protrusions on LM8 cells (right). Values are mean ± SEMs (≥30 cells per group). ** P

    Article Snippet: Anti-α-tubulin (DM1A, ab7291), anti-pericentrin (ab4448), and anti-Tyr397-phosphorylated FAK (p-FAK, EP2160Y, ab81298), Goat Anti-Mouse IgG (Alexa Fluor® 488, ab150113), Donkey Anti-Rabbit IgG (Alexa Fluor® 555, ab150062), and Donkey Anti-Mouse IgG (Alexa Fluor® 555, ab150110) were purchased from Abcam (Cambridge, UK).

    Techniques: Migration, Staining

    CG30463/pgant9 encodes a Golgi-localized O-glycosyltransferase. a Gene structure for CG30463/pgant9 is shown, with boxes representing exons and lines representing introns. The N-terminal (blue), catalytic (orange) and lectin (green) domains of the putative glycosyltransferase encoded by CG30463 are shown. The lectin domain consists of three subdomains (α, β, and γ). The sequence for the differentially spliced α subdomain (exon 8) is shown, with acidic residues highlighted in red and basic residues highlighted in blue. b Both splice variants (V5-tagged; red) localized to the Golgi apparatus (as detected by anti-GM130; blue) in S2R+ cells. Scale bar, 10 μm. Representative images from two independent experiments are shown. c Western blots of S2R+ cells expressing vector alone (Vector), a V5-tagged recombinant CG30463A or a V5-tagged recombinant CG30463B . Panels on the left show CG30463A and CG30463B expression with the V5-tag (anti-V5) and loading controls (anti-tubulin). Panel on the right shows increased O-glycosylation (as detected by the lectin HPA) when CG30463A or CG30463B are expressed in S2R+ cells. Representative western blots from three independent experiments are shown. Molecular weight markers (kD) are shown to the left of each panel

    Journal: Nature Communications

    Article Title: A molecular switch orchestrates enzyme specificity and secretory granule morphology

    doi: 10.1038/s41467-018-05978-9

    Figure Lengend Snippet: CG30463/pgant9 encodes a Golgi-localized O-glycosyltransferase. a Gene structure for CG30463/pgant9 is shown, with boxes representing exons and lines representing introns. The N-terminal (blue), catalytic (orange) and lectin (green) domains of the putative glycosyltransferase encoded by CG30463 are shown. The lectin domain consists of three subdomains (α, β, and γ). The sequence for the differentially spliced α subdomain (exon 8) is shown, with acidic residues highlighted in red and basic residues highlighted in blue. b Both splice variants (V5-tagged; red) localized to the Golgi apparatus (as detected by anti-GM130; blue) in S2R+ cells. Scale bar, 10 μm. Representative images from two independent experiments are shown. c Western blots of S2R+ cells expressing vector alone (Vector), a V5-tagged recombinant CG30463A or a V5-tagged recombinant CG30463B . Panels on the left show CG30463A and CG30463B expression with the V5-tag (anti-V5) and loading controls (anti-tubulin). Panel on the right shows increased O-glycosylation (as detected by the lectin HPA) when CG30463A or CG30463B are expressed in S2R+ cells. Representative western blots from three independent experiments are shown. Molecular weight markers (kD) are shown to the left of each panel

    Article Snippet: For immunoblotting, anti-V5-HRP (1:5000) (Invitrogen, #R961–25), anti-Tubulin (1:1000) (Cell Signaling Technology, #2125), and HRP-conjugated anti-rabbit antibody (1:2000) (Cell Signaling Technology, #7074) were used.

    Techniques: Sequencing, Western Blot, Expressing, Plasmid Preparation, Recombinant, Molecular Weight

    pgant9 undergoes tissue-specific splicing. a Expression of each splice variant was quantitated in various larval tissues by qPCR. pgant9B expression is most abundant in the salivary gland while pgant9A is the predominant isoform in other tissues examined. RNA levels were normalized to 18S rRNA. Values represent mean ± s.d. from four experiments. b Western blots of salivary gland extracts from WT larvae or larvae expressing RNAi to pgant9 ( pgant9 RNAi ) probed with a lectin (peanut agglutinin; PNA) that detects the major salivary gland O-glycans. Size shifts in the three major PNA-reactive bands are seen (denoted with arrows on the right side of the panel). Tubulin loading control is shown in the lower panel. Representative western blot from four independent experiments are shown. Size markers are shown to the left of each panel

    Journal: Nature Communications

    Article Title: A molecular switch orchestrates enzyme specificity and secretory granule morphology

    doi: 10.1038/s41467-018-05978-9

    Figure Lengend Snippet: pgant9 undergoes tissue-specific splicing. a Expression of each splice variant was quantitated in various larval tissues by qPCR. pgant9B expression is most abundant in the salivary gland while pgant9A is the predominant isoform in other tissues examined. RNA levels were normalized to 18S rRNA. Values represent mean ± s.d. from four experiments. b Western blots of salivary gland extracts from WT larvae or larvae expressing RNAi to pgant9 ( pgant9 RNAi ) probed with a lectin (peanut agglutinin; PNA) that detects the major salivary gland O-glycans. Size shifts in the three major PNA-reactive bands are seen (denoted with arrows on the right side of the panel). Tubulin loading control is shown in the lower panel. Representative western blot from four independent experiments are shown. Size markers are shown to the left of each panel

    Article Snippet: For immunoblotting, anti-V5-HRP (1:5000) (Invitrogen, #R961–25), anti-Tubulin (1:1000) (Cell Signaling Technology, #2125), and HRP-conjugated anti-rabbit antibody (1:2000) (Cell Signaling Technology, #7074) were used.

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

    Mutations in MNS1 when combined with mutations in DNAH5 might result in defects of the ODA-microtubule docking complex in human respiratory epithelial cells. (A) Transmission electron micrographs show subtle ultrastructural defects in affected individual AL-III-9 carrying bi-allelic MNS1 mutations with the occasional absence of only few ODAs (2–4 out of 9) in about half of the cross-sections (compared to control samples where all analyzed sections show an average of 8.7 ODAs, 9 analyzed sections from the MNS1-deficient ciliary axonemes show an average of 6 ODAs). However, TEM show complete absence of ODAs in PCD-affected individuals OI-24 II1 ( DNAH5 mutations) and OI-11 II6 ( MNS1 and DNAH5 mutations) compared to a control without PCD. In the healthy control, outer dynein arms are visible (blue arrows). However, the cilia from OI-24 II1 still have the ODA-DC (small projections marked by white arrows) whereas the cilia from OI-11 II6 do not, suggesting that MNS1 deficiency when combined with DNAH5 deficiency might cause defects in ODA-DC assembly. Below the control TEM section a schematic illustrating a microtubular doublet with attached ODA docking complex (ODA-DC) and the double-headed ODA complex proteins with dynein heavy chain DNAH5 and dynein intermediate chains DNAI1 and DNAI2. In affected individual AL-III-9, a partial defect is observed; in OI-24II1, a schematic where the ODA complex is absent while the ODA-DC is still retained; in OI-11II6, a schematic where both ODA and ODA docking complexes are absent. Scale bars, 0.1 μm. (B) Respiratory epithelial cells from control and affected individuals: AL-III-9 carrying bi-allelic MNS1 mutations, OI-11 II6 carrying bi-allelic MNS1 and DNAH5 mutations and OI-24 II1 carrying no mutations in MNS1 but identical bi-allelic DNAH5 mutations as OI-11 II6. For space issues, OI-24 II1 is described as DNAH5 mut/mut instead of DNAH5 c . 13432_13435delCACT/ c . 13432_13435delCACT . Cells were double-labeled with antibodies directed against acetylated alpha-tubulin (green) and CCDC114 (HPA042524, Atlas antibodies) (red). Nuclei were stained with Hoechst 33342 (blue). Both proteins co-localize (yellow) along the ciliary axonemes in cells from the unaffected controls, AL-III-9 and OI-24 II1, while in cells of OI-11 II6, CCDC114 localizes only to the proximal part of the ciliary axonemes, indicating that recessive loss-of-function mutations in MNS1 when combined with loss-of-function mutations in DNAH5 might affect the distal localization of ODA-DC associated proteins and might play a role in docking or anchoring the ODA subunits or in regulating this process. Scale bars, 10μm.

    Journal: PLoS Genetics

    Article Title: Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility

    doi: 10.1371/journal.pgen.1007602

    Figure Lengend Snippet: Mutations in MNS1 when combined with mutations in DNAH5 might result in defects of the ODA-microtubule docking complex in human respiratory epithelial cells. (A) Transmission electron micrographs show subtle ultrastructural defects in affected individual AL-III-9 carrying bi-allelic MNS1 mutations with the occasional absence of only few ODAs (2–4 out of 9) in about half of the cross-sections (compared to control samples where all analyzed sections show an average of 8.7 ODAs, 9 analyzed sections from the MNS1-deficient ciliary axonemes show an average of 6 ODAs). However, TEM show complete absence of ODAs in PCD-affected individuals OI-24 II1 ( DNAH5 mutations) and OI-11 II6 ( MNS1 and DNAH5 mutations) compared to a control without PCD. In the healthy control, outer dynein arms are visible (blue arrows). However, the cilia from OI-24 II1 still have the ODA-DC (small projections marked by white arrows) whereas the cilia from OI-11 II6 do not, suggesting that MNS1 deficiency when combined with DNAH5 deficiency might cause defects in ODA-DC assembly. Below the control TEM section a schematic illustrating a microtubular doublet with attached ODA docking complex (ODA-DC) and the double-headed ODA complex proteins with dynein heavy chain DNAH5 and dynein intermediate chains DNAI1 and DNAI2. In affected individual AL-III-9, a partial defect is observed; in OI-24II1, a schematic where the ODA complex is absent while the ODA-DC is still retained; in OI-11II6, a schematic where both ODA and ODA docking complexes are absent. Scale bars, 0.1 μm. (B) Respiratory epithelial cells from control and affected individuals: AL-III-9 carrying bi-allelic MNS1 mutations, OI-11 II6 carrying bi-allelic MNS1 and DNAH5 mutations and OI-24 II1 carrying no mutations in MNS1 but identical bi-allelic DNAH5 mutations as OI-11 II6. For space issues, OI-24 II1 is described as DNAH5 mut/mut instead of DNAH5 c . 13432_13435delCACT/ c . 13432_13435delCACT . Cells were double-labeled with antibodies directed against acetylated alpha-tubulin (green) and CCDC114 (HPA042524, Atlas antibodies) (red). Nuclei were stained with Hoechst 33342 (blue). Both proteins co-localize (yellow) along the ciliary axonemes in cells from the unaffected controls, AL-III-9 and OI-24 II1, while in cells of OI-11 II6, CCDC114 localizes only to the proximal part of the ciliary axonemes, indicating that recessive loss-of-function mutations in MNS1 when combined with loss-of-function mutations in DNAH5 might affect the distal localization of ODA-DC associated proteins and might play a role in docking or anchoring the ODA subunits or in regulating this process. Scale bars, 10μm.

    Article Snippet: Monoclonal mouse anti acetylated-α-tubulin (T7451) was obtained from Sigma (Germany).

    Techniques: Transmission Assay, Transmission Electron Microscopy, Labeling, Staining

    Identification of MNS1 loss-of-function mutations in a PCD-affected individual with DNAH5 mutations. (A) Pedigree of families OI-11, OI-14 and OI-24: consanguinity of first degree. In total, five PCD-affected individuals carry homozygous mutations in DNAH5 (annotated as DNAH5 mut/mut ) of whom only one (OI-11 II6) carries additional homozygous mutations in MNS1 (c.607C > T; p.Gln203*). (B) Bi-allelic MNS1 nonsense mutations in OI-11 II6 (c.607C > T) predicting a premature termination of translation (p.Gln203*). The affected sibling OI-11 II1 and both parents OI-11 I1 and I2 are carriers of the mutant allele. Right panel . Bi-allelic DNAH5 nonsense mutations in OI-11 II6 and OI-11 II1 (c. c.13432_13435delCACT) predicting a premature termination of translation (p.His4478Alafs3*). Both parents OI-11 I1 and I2 are carriers of the mutant allele. (C) Respiratory epithelial cells from control and affected individuals: OI-11 II6 carrying bi-allelic MNS1 and DNAH5 mutations, and OI-24 II1 carrying the identical bi-allelic DNAH5 mutations as OI-11 II6. For space issues, OI-24 II1 is referred to in this and other Figures as DNAH5 mut/mut instead of DNAH5 c . 13432_13435delCACT/ c . 13432_13435delCACT . Cells were double-labeled with antibodies directed against acetylated alpha-tubulin (green) and MNS1 (red). Nuclei were stained with Hoechst 33342 (blue). Both proteins co-localize (yellow) along the ciliary axonemes in cells from the unaffected controls and OI-24 II1, while in respiratory cells of OI-11 II6, MNS1 is undetectable in the ciliary axonemes, consistent with recessive loss-of-function MNS1 nonsense mutations. Scale bars, 10μm.

    Journal: PLoS Genetics

    Article Title: Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility

    doi: 10.1371/journal.pgen.1007602

    Figure Lengend Snippet: Identification of MNS1 loss-of-function mutations in a PCD-affected individual with DNAH5 mutations. (A) Pedigree of families OI-11, OI-14 and OI-24: consanguinity of first degree. In total, five PCD-affected individuals carry homozygous mutations in DNAH5 (annotated as DNAH5 mut/mut ) of whom only one (OI-11 II6) carries additional homozygous mutations in MNS1 (c.607C > T; p.Gln203*). (B) Bi-allelic MNS1 nonsense mutations in OI-11 II6 (c.607C > T) predicting a premature termination of translation (p.Gln203*). The affected sibling OI-11 II1 and both parents OI-11 I1 and I2 are carriers of the mutant allele. Right panel . Bi-allelic DNAH5 nonsense mutations in OI-11 II6 and OI-11 II1 (c. c.13432_13435delCACT) predicting a premature termination of translation (p.His4478Alafs3*). Both parents OI-11 I1 and I2 are carriers of the mutant allele. (C) Respiratory epithelial cells from control and affected individuals: OI-11 II6 carrying bi-allelic MNS1 and DNAH5 mutations, and OI-24 II1 carrying the identical bi-allelic DNAH5 mutations as OI-11 II6. For space issues, OI-24 II1 is referred to in this and other Figures as DNAH5 mut/mut instead of DNAH5 c . 13432_13435delCACT/ c . 13432_13435delCACT . Cells were double-labeled with antibodies directed against acetylated alpha-tubulin (green) and MNS1 (red). Nuclei were stained with Hoechst 33342 (blue). Both proteins co-localize (yellow) along the ciliary axonemes in cells from the unaffected controls and OI-24 II1, while in respiratory cells of OI-11 II6, MNS1 is undetectable in the ciliary axonemes, consistent with recessive loss-of-function MNS1 nonsense mutations. Scale bars, 10μm.

    Article Snippet: Monoclonal mouse anti acetylated-α-tubulin (T7451) was obtained from Sigma (Germany).

    Techniques: Mutagenesis, Labeling, Staining

    MNS1 localizes to human respiratory cilia and human sperm flagella. (A) Western blot analysis of protein lysates from human respiratory cells (M, protein standard). MNS1 antibodies specifically detect a single band with the predicted size (~61kDa, lane 1). (B) Respiratory epithelial cells from control and affected individual AL-III-9 carrying bi-allelic MNS1 mutations. Cells were double-labeled with antibodies directed against acetylated alpha-tubulin (green) and MNS1 (red). Nuclei were stained with Hoechst 33342 (blue). Both proteins co-localize (yellow) along the ciliary axonemes in cells from the unaffected controls, while in respiratory cells of AL-III-9, MNS1 is undetectable in the ciliary axonemes, consistent with recessive loss-of-function MNS1 nonsense mutations. Scale bars, 10μm. (C) Western blot analysis of lysate from human whole sperm cells (M, protein standard). Anti-MNS1 antibody specifically detects a band at the predicted size (~61kDa, lane 1) and a band of approximately ~45kDa, indicating an isoform of MNS1 in sperm. (D) In human control spermatozoa, MNS1 (red) co-localizes with acetylated alpha-tubulin (green) along flagellar axonemes except at the endpiece (white box).

    Journal: PLoS Genetics

    Article Title: Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility

    doi: 10.1371/journal.pgen.1007602

    Figure Lengend Snippet: MNS1 localizes to human respiratory cilia and human sperm flagella. (A) Western blot analysis of protein lysates from human respiratory cells (M, protein standard). MNS1 antibodies specifically detect a single band with the predicted size (~61kDa, lane 1). (B) Respiratory epithelial cells from control and affected individual AL-III-9 carrying bi-allelic MNS1 mutations. Cells were double-labeled with antibodies directed against acetylated alpha-tubulin (green) and MNS1 (red). Nuclei were stained with Hoechst 33342 (blue). Both proteins co-localize (yellow) along the ciliary axonemes in cells from the unaffected controls, while in respiratory cells of AL-III-9, MNS1 is undetectable in the ciliary axonemes, consistent with recessive loss-of-function MNS1 nonsense mutations. Scale bars, 10μm. (C) Western blot analysis of lysate from human whole sperm cells (M, protein standard). Anti-MNS1 antibody specifically detects a band at the predicted size (~61kDa, lane 1) and a band of approximately ~45kDa, indicating an isoform of MNS1 in sperm. (D) In human control spermatozoa, MNS1 (red) co-localizes with acetylated alpha-tubulin (green) along flagellar axonemes except at the endpiece (white box).

    Article Snippet: Monoclonal mouse anti acetylated-α-tubulin (T7451) was obtained from Sigma (Germany).

    Techniques: Western Blot, Labeling, Staining