anti-α-tubulin Search Results


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  • 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
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    Millipore α tubulin
    Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. <t>α-tubulin</t> is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *
    α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 22359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    99
    Millipore mouse 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).
    Mouse Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc 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).
    α Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 7202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    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
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    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
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    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
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    99
    Abcam α 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
    α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti tubulin acetylated antibody
    Mounting samples for visualization of Kupffer’s vesicle. Schematic showing a 10 somite stage zebrafish embryo ( A ). Forceps can be used as depicted to separate the tail tip from the rest of the embryo. Excess yolk is then removed, and the tail tip mounted flat on to a microscope slide. ( B ) Bright field image overlaid with fluorescence image of flat-mounted tail tip. The white box depicts the location of Kupffer’s vesicle. Fluorescence from acetylated-α <t>tubulin</t> (red) can be seen within this region. ( C ) Confocal image of Kupffer’s vesicle, visualised using acetylated-α tubulin (red), aPKC (green) and DAPI (blue).
    Monoclonal Anti Tubulin Acetylated Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3808 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Image Search Results


    Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. α-tubulin is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: Acute expression of F3-T3 fusion induces peroxisome biogenesis through phosphorylation of Y122 of PIN4 a , Representative confocal microphotographs (maximum intensity) of immunofluorescence staining for total PIN4 (PIN4, red, left panel) and pY122-PIN4 (pPIN4, red, middle panel) in vector and HA expressing F3-T3. Right panels show higher magnification of dotted boxes. Nuclei were counterstained with DAPI (blue). Experiment was repeated independently two times with similar results. b , Maximum intensity of confocal images of double immunofluorescence staining for FGFR3 (green, middle panel) and pY122-PIN4 (red, right panel) in HA-F3-T3. Arrows indicate protein co-localization. Experiment was repeated independently two times with similar results. c , Co-immunoprecipitation from H1299 cells using PIN4 antibody. Endogenous PIN4 immunocomplexes and input (WCL) were analyzed by western blot using the indicated antibodies. Input is 10% for PEX1, PEX6, SUN2 and NUP214; 5% for SEC16A and DHX30; 2% for PIN4. d , Western blot analysis of co-immunoprecipitation of exogenous FLAG-PEX1 in HA-F3-T3. WCL: 1% for PIN4 and 10% for PEX1 and PEX6. Experiment was repeated independently four times with similar results. e , qRT-PCR for PEX1 in HA-F3-T3 and HA-vector. Data are Mean±s.d. (n=3 technical replicates) of one representative experiment out of three independent experiments performed in triplicate. f , Western blot analysis of PEX1 expression in HA transduced with F3-T3, F3-T3-K508M or the empty vector. β-actin is shown as loading control. Experiment was repeated independently three times with similar results. g , Time course analysis of F3-T3 expression in HA by western blot. α-tubulin is shown as loading control. Experiment was repeated independently two times with similar results. h , Quantification of protein biosynthesis by OPP incorporation measured by high-content fluorescent microscopy in HA reconstituted with PIN4-WT or PIN4-Y122F after silencing of the endogenous PIN4 and acutely transduced with F3-T3 or vector. Representative bar plots (n=4 technical replicates) from one out of three independent experiments. P: *

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Expressing, Immunofluorescence, Staining, Plasmid Preparation, Double Immunofluorescence Staining, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Transduction, Microscopy

    Functional analysis of tyrosine phosphorylation of F3-T3 kinase substrates a , Western blot analysis of phosphotyrosine immunoprecipitation of mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53 using PIN4 antibody. F3-T3 and RAS-12V expression are shown. α-tubulin is shown as loading control b , Microphotographs of immunofluorescence using the pY122-PIN4 specific antibody (red, upper panels) in tumors from mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53. Nuclei were counterstained with DAPI (blue, lower panels). Experiment was repeated independently two times with similar results. c , Representative microphotographs of pY122-PIN4 immunofluorescence in F3-T3-positive (upper panels) and F3-T3-negative (lower panels) GBM (green). Right panels show higher magnification of pY122-PIN4-DAPI co-staining depicting cytoplasmic localization of pY122-PIN4. DAPI staining of nuclei is shown as an indication of cellular density (middle panels). d , Analysis of OCR of HA-F3-T3 transduced with WT, or the unphosphorylable mutant (Y/A) of GOLGIN84, C1ORF50 and DLG3. HA-vector are included as control. Data are Mean±s.d. (n=5 technical replicates) of one representative experiment out of two independent experiments performed in triplicate with similar results. P

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: Functional analysis of tyrosine phosphorylation of F3-T3 kinase substrates a , Western blot analysis of phosphotyrosine immunoprecipitation of mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53 using PIN4 antibody. F3-T3 and RAS-12V expression are shown. α-tubulin is shown as loading control b , Microphotographs of immunofluorescence using the pY122-PIN4 specific antibody (red, upper panels) in tumors from mGSC-F3-T3-sh TP53 and mGSC-HRAS-12V-sh TP53. Nuclei were counterstained with DAPI (blue, lower panels). Experiment was repeated independently two times with similar results. c , Representative microphotographs of pY122-PIN4 immunofluorescence in F3-T3-positive (upper panels) and F3-T3-negative (lower panels) GBM (green). Right panels show higher magnification of pY122-PIN4-DAPI co-staining depicting cytoplasmic localization of pY122-PIN4. DAPI staining of nuclei is shown as an indication of cellular density (middle panels). d , Analysis of OCR of HA-F3-T3 transduced with WT, or the unphosphorylable mutant (Y/A) of GOLGIN84, C1ORF50 and DLG3. HA-vector are included as control. Data are Mean±s.d. (n=5 technical replicates) of one representative experiment out of two independent experiments performed in triplicate with similar results. P

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Functional Assay, Western Blot, Immunoprecipitation, Expressing, Immunofluorescence, Staining, Transduction, Mutagenesis, Plasmid Preparation

    F3-T3 induces sensitivity to inhibitors of mitochondrial metabolism a , Immunoblot analysis using the FGFR3 antibody in HA-vector, HA-F3-T3 or HA-F3-T3-K508M. α-tubulin is shown as loading control. Experiment was repeated five times with similar results. b , OCR of GSC1123 harboring F3-T3 in the presence or absence of AZD4547. Data are Mean±s.d. (n=6 technical replicates) of one representative experiment out of two independent experiments. P:

    Journal: Nature

    Article Title: A metabolic function of FGFR3-TACC3 gene fusions in cancer

    doi: 10.1038/nature25171

    Figure Lengend Snippet: F3-T3 induces sensitivity to inhibitors of mitochondrial metabolism a , Immunoblot analysis using the FGFR3 antibody in HA-vector, HA-F3-T3 or HA-F3-T3-K508M. α-tubulin is shown as loading control. Experiment was repeated five times with similar results. b , OCR of GSC1123 harboring F3-T3 in the presence or absence of AZD4547. Data are Mean±s.d. (n=6 technical replicates) of one representative experiment out of two independent experiments. P:

    Article Snippet: Antibodies and concentrations are: FGFR3 1:1000 (Santa Cruz, B-9, sc-13121), PIN4 1:1000 (Abcam, ab155283), PKM2 1:1000 (Cell Signaling, #3198), DLG3/SAP102 1:1000 (Cell Signaling, #3733), GOLGIN84 1:2000 (Santa Cruz, H-283, sc-134704); C1ORF50 1:1000 (Novus Biologicals, NBP1-81053), HGS 1:1000 (Abcam, ab72053), FAK 1:1000 (Cell Signaling, 3285), Paxillin 1:1000 (BD Transduction, 610051), PGC1α 1:500 (Santa Cruz, H300, sc-13067), PGC1α 1:1000 (Novus Biological, NBP104676), ESRRG 1:500 (Abcam, ab128930), ESRRG 1:500 (R7D, PP-H6812000) p-FRS2 1:1000 (Cell Signaling, 3861), FRS2 1:1000 (Santa Cruz, sc-8318), p-STAT3 1:1000 (Cell Signaling, #9131), STAT3 1:1000 (Santa Cruz, C-20 sc-482,), p-AKT 1:1000 (Cell Signaling, #4060), AKT 1:1000 (Cell Signaling, #9272), p-ERK1/2 1:1000 (Cell Signaling, #4370), ERK1/2 1:1000 (Cell Signaling, #9102), β-actin 1:2000 (Sigma, A5441), PEX1 1:500 (BD Biosciences #611719), PEX6 1:500 (Stress Marq, #SMC-470), NUP214 1:500 (Abcam #ab70497), SEC16A 1:500 (Abcam #ab70722), DHX30 1:500 (Novus Biologicals, NBP1-26203), SUN-2 1:500 (Abcam #ab124916), FLAG 1:1000 (Abcam ab1162), Retinoblastoma 1:1000 (BD Pharmingen 554136), α-tubulin 1:2000 (Sigma, T5168), total OXPHOS 1:1000 (Abcam, #ab110411), MTCO1 1:1000 (Abcam, #ab14705).

    Techniques: Plasmid Preparation

    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

    GBM-SKH cells were stably expressed without or with the control vector (pcDNA3.1) or with wild-type OPN (pDNA3.1/OPN). (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were then collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts of experimental cells in the upper part of a transwell chamber separated by a Matrigel-coated membrane. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with GBM/pcDNA or GBM/OPN were evaluated. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: GBM-SKH cells were stably expressed without or with the control vector (pcDNA3.1) or with wild-type OPN (pDNA3.1/OPN). (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were then collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts of experimental cells in the upper part of a transwell chamber separated by a Matrigel-coated membrane. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with GBM/pcDNA or GBM/OPN were evaluated. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Article Snippet: Anti-OPN and anti-α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California).

    Techniques: Stable Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Activity Assay, In Vitro, Invasion Assay

    U87MG cells were either stably transfected with an OPN shRNA plasmid or with a plKo shRNA vector as mock control. (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts cells in the upper part of transwell chamber separated by a Matrigel-coated membrane. Migrated cells on the bottom of the membrane were counted. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with U87/plKo or U87MG/shOPN. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: U87MG cells were either stably transfected with an OPN shRNA plasmid or with a plKo shRNA vector as mock control. (A) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (B) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, and α-tubulin. These experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to mock control cells. Results shown in the lower panel of (A) and (B) are the mean ± SE of three independent experiments. (C) Media were collected for the zymographic assay of MMP-2 activity. In the lower panel, MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (D) For the in vitro invasion assay, cells were seeded in equal amounts cells in the upper part of transwell chamber separated by a Matrigel-coated membrane. Migrated cells on the bottom of the membrane were counted. Data represent the mean ± SE of three independent experiments. (E) Cells were seeded in culture plates in DMEM media supplemented with 10% heat-inactivated FCS. Viable cell numbers were counted. Data represent the mean ± SE of three independent experiments. (F) Histopathological characteristics (H E) and tumor volumes of animals implanted with U87/plKo or U87MG/shOPN. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Article Snippet: Anti-OPN and anti-α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California).

    Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Activity Assay, In Vitro, Invasion Assay

    Cell lines derived from human gliomas exhibit different growth rates, invasiveness, and intravasation. (A) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel. After 24 hours, cells on the bottom side of filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (B) Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (C) A chicken embryo CAM model was used for the in vivo intravasation assay. Intravasation was demonstrated by amplification of the human Alu sequence from DNA extracted from the isolated CAMs. P, positive control; N, negative control. (D) Medium was collected and MMP-2 gelatinolytic activities were analyzed by zymography. In the lower panel, the MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (E) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (F) Cell lysates were harvested and immunoblotted with antibodies specific for MMP-2, OPN, and α-tubulin. The upper panels of (E) and (F) are representative data, these experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to equal loading controls. Results shown in the lower panels are the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: Cell lines derived from human gliomas exhibit different growth rates, invasiveness, and intravasation. (A) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel. After 24 hours, cells on the bottom side of filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (B) Viable cell numbers were counted at 24, 48, and 72 hours. Data represent the mean ± SE of three independent experiments. (C) A chicken embryo CAM model was used for the in vivo intravasation assay. Intravasation was demonstrated by amplification of the human Alu sequence from DNA extracted from the isolated CAMs. P, positive control; N, negative control. (D) Medium was collected and MMP-2 gelatinolytic activities were analyzed by zymography. In the lower panel, the MMP-2 activity was quantitated by scanning the photographic negatives on a gel analysis system. (E) MMP-2, OPN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expressions were analyzed by RT–PCR. (F) Cell lysates were harvested and immunoblotted with antibodies specific for MMP-2, OPN, and α-tubulin. The upper panels of (E) and (F) are representative data, these experiments were repeated, and the intensities of bands were quantitated and expressed as ratios to equal loading controls. Results shown in the lower panels are the mean ± SE of three independent experiments. * P

    Article Snippet: Anti-OPN and anti-α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California).

    Techniques: Derivative Assay, In Vitro, Invasion Assay, Staining, Chick Chorioallantoic Membrane Assay, In Vivo, Amplification, Sequencing, Isolation, Positive Control, Negative Control, Zymography, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    Human glioma cell lines were treated with different concentrations of 5-aza-dC. (A) Media were then collected for a zymographic assay for MMP-2 activity. In the lower panel, effects of 5-aza-dC on MMP-2 secretion in various cells were quantitated by scanning the photographic negatives on a gel analysis system and are expressed as a percent of basal level for each cell line. (B) Cell viability was analyzed by the MTT assay after incubation of U87MG cells with 5-aza-dC for 24 hours. (C) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, GFAP, vimentin, and α-tubulin. (D) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel in the presence of different concentrations of 5-aza-dC. After 24 hours, cells on the bottom side of the filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (E) An intravasation assay was carried out. PCR amplification for Alu sequence is shown. P, positive control; N, negative control. (F) A trypan blue exclusion assay was carried out after treating cells with different concentrations of 5-aza-dC in 5% FBS medium for different time periods. Data represent the mean ± SE of three independent experiments. (G) Measurement of implanted tumor sizes in mice without or with 5-aza-dC administration. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Journal: Neuro-Oncology

    Article Title: Osteopontin regulates human glioma cell invasiveness and tumor growth in mice

    doi: 10.1093/neuonc/nop013

    Figure Lengend Snippet: Human glioma cell lines were treated with different concentrations of 5-aza-dC. (A) Media were then collected for a zymographic assay for MMP-2 activity. In the lower panel, effects of 5-aza-dC on MMP-2 secretion in various cells were quantitated by scanning the photographic negatives on a gel analysis system and are expressed as a percent of basal level for each cell line. (B) Cell viability was analyzed by the MTT assay after incubation of U87MG cells with 5-aza-dC for 24 hours. (C) Cell lysates were harvested and immunoblotted with antibodies specific for OPN, MMP-2, GFAP, vimentin, and α-tubulin. (D) For the in vitro invasion assay, equal numbers of glioma cells were seeded in the upper part of a transwell coated with Matrigel in the presence of different concentrations of 5-aza-dC. After 24 hours, cells on the bottom side of the filter were fixed, stained, and counted. Data represent the mean ± SE of three independent experiments. (E) An intravasation assay was carried out. PCR amplification for Alu sequence is shown. P, positive control; N, negative control. (F) A trypan blue exclusion assay was carried out after treating cells with different concentrations of 5-aza-dC in 5% FBS medium for different time periods. Data represent the mean ± SE of three independent experiments. (G) Measurement of implanted tumor sizes in mice without or with 5-aza-dC administration. Tumor sizes were measured through their largest diameter (upper panel), and the lower panel shows the mean ± SE of three independent experiments. * P

    Article Snippet: Anti-OPN and anti-α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California).

    Techniques: Activity Assay, MTT Assay, Incubation, In Vitro, Invasion Assay, Staining, Polymerase Chain Reaction, Amplification, Sequencing, Positive Control, Negative Control, Trypan Blue Exclusion Assay, Mouse Assay

    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

    Mounting samples for visualization of Kupffer’s vesicle. Schematic showing a 10 somite stage zebrafish embryo ( A ). Forceps can be used as depicted to separate the tail tip from the rest of the embryo. Excess yolk is then removed, and the tail tip mounted flat on to a microscope slide. ( B ) Bright field image overlaid with fluorescence image of flat-mounted tail tip. The white box depicts the location of Kupffer’s vesicle. Fluorescence from acetylated-α tubulin (red) can be seen within this region. ( C ) Confocal image of Kupffer’s vesicle, visualised using acetylated-α tubulin (red), aPKC (green) and DAPI (blue).

    Journal: F1000Research

    Article Title: Using zebrafish to study the function of nephronophthisis and related ciliopathy genes

    doi: 10.12688/f1000research.15511.1

    Figure Lengend Snippet: Mounting samples for visualization of Kupffer’s vesicle. Schematic showing a 10 somite stage zebrafish embryo ( A ). Forceps can be used as depicted to separate the tail tip from the rest of the embryo. Excess yolk is then removed, and the tail tip mounted flat on to a microscope slide. ( B ) Bright field image overlaid with fluorescence image of flat-mounted tail tip. The white box depicts the location of Kupffer’s vesicle. Fluorescence from acetylated-α tubulin (red) can be seen within this region. ( C ) Confocal image of Kupffer’s vesicle, visualised using acetylated-α tubulin (red), aPKC (green) and DAPI (blue).

    Article Snippet: For simple detection of cilia structure the best antibody is anti-acetylated-α tubulin (Sigma-Aldrich; catalogue number T6793) at 1:500 for the ciliary axoneme.

    Techniques: Microscopy, Fluorescence

    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