human α-tubulin Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore monoclonal anti alpha tubulin antibody
    Monoclonal Anti Alpha Tubulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti alpha tubulin antibody/product/Millipore
    Average 99 stars, based on 19851 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti alpha tubulin antibody - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Millipore human α tubulin
    Histochemistry analysis of brain sections from patient AD1. Evidence of CA in the brain cortex containing microbial proteins. Insula cortex (INCO) sections from patient AD1 stained with Periodic acid–Schiff reagent (upper panel); immunostained with a rabbit polyclonal anti- C. albicans antibody (green) used at 1:100 dilution, and a mouse monoclonal anti-human <t>α-tubulin</t> antibody (red) used at 1:50 dilution (middle panel); immunostained with a mouse monoclonal anti-peptidoglycan antibody (green) used at 1:20 dilution, and a rabbit polyclonal anti- C . albicans antibody (red) used at 1:100 dilution (lower panel). DAPI staining of nuclei appears in blue. Scale bar: 50 μm.
    Human α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human α tubulin/product/Millipore
    Average 99 stars, based on 860 article reviews
    Price from $9.99 to $1999.99
    human α tubulin - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    94
    Cedarlane anti human alpha tubulin ascites clone dm1a mouse igg1
    Inflammation induces EndoMT in BECs. a Confluent BECs were stimulated with TGF-β1 and IL-1β as described in the materials and method section, mRNA expression levels of different EndoMT-associated TFs, and endothelial or mesenchymal markers were determined by qRT-PCR. Values were normalized to GAPDH and plotted as fold change relative to control (dotted line). b – f TGF-β1- and IL-1β-stimulated BECs total and nuclear protein levels of SNAI1, CLDN5, CDH5, and CDH2 was assessed by western blot and expressed as fold change compared to control cells. Values were normalized to β-actin, <t>α-tubulin</t> or Lamin B levels. g – p CLDN5, CDH5, OCLN, CDH2, and FSP1 protein expression was assessed by immunofluorescence and imaged under the confocal microscope (scale bar 20 µm). Data presented are the mean of triplicate values ± SEM of three independent experiments. Statistical analysis was performed using Student’s t -test or one-way ANOVA, where * p
    Anti Human Alpha Tubulin Ascites Clone Dm1a Mouse Igg1, supplied by Cedarlane, used in various techniques. Bioz Stars score: 94/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human alpha tubulin ascites clone dm1a mouse igg1/product/Cedarlane
    Average 94 stars, based on 161 article reviews
    Price from $9.99 to $1999.99
    anti human alpha tubulin ascites clone dm1a mouse igg1 - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    95
    R&D Systems α tubulin
    Increased hepatic IP-10 expression and liver homing of NK cells following iNKT cell activation. Livers of wt mice were excised 12 h after s.c. administration of a single dose of αGalCerMPEG (10 µg) and prepared for histopathological or Western blot analysis. ( A – F ) histopathological analysis of IP-10 expression (brown) in the liver of untreated ( A – C ) and treated ( D – F ) mice at an original magnification of x10 ( A , D ) and x20 ( B , C and E , F ). ( G ) Western blot analysis of monomeric IP-10 expression in hepatocytes isolated from untreated and αGalCerMPEG- administered mice (n = 9–11 mice, shown is one out of at least three independent experiments). The data are presented as the ratio of the volume of integrals of the signals detected for IP-10 and <t>α-tubulin.</t> For the in vivo migration studies, CD45.1 + NK cells were adoptively transferred intravenously (i.v.) into wt mice. Recipient mice were administered s.c. a single dose of αGalCerMPEG (10 µg) 24 h later and lymphocytes derived from spleen and liver were analyzed 72 h after treatment. Frequencies and absolute numbers of CD3 − NK1.1 + CD45.1 + cells (out of 1 × 10 6 total cells) derived from ( H ) the spleen and ( I ) the liver of untreated (white) and treated (black) mice. Shown are the compiled data derived from two independent experiments (n = 7–8 mice). ( J ) The mechanism of NK cell migration was assessed using a trans-well system. Hepatocytes, serum and NK cells were obtained from untreated (−) or treated (+) mice 12 h after administration of a single dose of αGalCerMPEG (s.c., 10 µg). Absolute numbers of CFSE + NK cells (out of 1 × 10 6 total cells) were assessed after 2 h incubation at 37 °C (n = 3 mice, shown is one out of two independent experiments). Columns represent the mean ± SEM and circles indicate single values. Violin plots represent the interquartile range, horizontal lines show the mean value and the width displays the distribution of data points. Asterisks denote significant values as calculated by unpaired, two-tailed Student’s t-test as compared to untreated control. **p ≤ 0.01, *p ≤ 0.05, n.s. = not significant.
    α Tubulin, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α tubulin/product/R&D Systems
    Average 95 stars, based on 46 article reviews
    Price from $9.99 to $1999.99
    α tubulin - by Bioz Stars, 2020-11
    95/100 stars
      Buy from Supplier

    89
    Santa Cruz Biotechnology mouse anti human α tubulin
    The expression of CD74 and CD44 receptors in immortalized normal breast luminal cells (226LDM) (A) Cell-surface and intracellular expression of CD74 and CD44 was acquired by flow cytometry using By2 (anti-CD74) and 156-3C11 (anti-CD44). Black-filled histograms represent the 226LDM cells stained with indicated antibody. Empty histograms show the isotype as negative controls. (B) Total protein of CD74 and CD44 was detected by Western blotting, and <t>α-tubulin</t> was used as a loading control. (C) Confocal images of CD74 and CD44 in 226LDM cells stained intracellularly: CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). Figures depict representative samples from duplicate experiments. Scale bar 10 μm.
    Mouse Anti Human α Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human α tubulin/product/Santa Cruz Biotechnology
    Average 89 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    mouse anti human α tubulin - by Bioz Stars, 2020-11
    89/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology human α tubulin
    C14orf166 upregulation in bladder cancer tissues. a Quantitative RT-PCR determination of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues from six patients. Transcription levels were normalized to GAPDH expression. b Western blot analysis of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues. <t>α-Tubulin</t> was used as the loading control. c IHC determination of C14orf166 expression in different patients with different clinical stages of disease according to T classification (magnification: ×200, ×400)
    Human α Tubulin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human α tubulin/product/Santa Cruz Biotechnology
    Average 91 stars, based on 43 article reviews
    Price from $9.99 to $1999.99
    human α tubulin - by Bioz Stars, 2020-11
    91/100 stars
      Buy from Supplier

    99
    Abcam mouse anti human α tubulin
    C14orf166 upregulation in bladder cancer tissues. a Quantitative RT-PCR determination of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues from six patients. Transcription levels were normalized to GAPDH expression. b Western blot analysis of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues. <t>α-Tubulin</t> was used as the loading control. c IHC determination of C14orf166 expression in different patients with different clinical stages of disease according to T classification (magnification: ×200, ×400)
    Mouse Anti Human α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human α tubulin/product/Abcam
    Average 99 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    mouse anti human α tubulin - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc mouse anti human α tubulin
    ZLN005 upregulates PGC-1 α and its downstream mitochondrial gene targets in ARPE-19 cells. (a) Relative gene expression (RE) of PGC-1α in ARPE-19 treated for 48 hours with 10 μ M ZLN005 (ZLN, n = 5) compared to vehicle (veh, n = 4). (b) Representative protein blot of PGC-1 α and <t>α</t> -tubulin bands and (c) quantification showed upregulation of PGC-1 α protein expression with ZLN005 treatment (all conditions, n = 4). (d) RPE-specific genes, BEST1 and RLBP1 , are upregulated with ZLN005 treatment (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (e) Treatment with 10 μ M ZLN005 increases the mitochondrial dynamic gene, MFN1 , and replication genes, TFAM and POLG (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (f, g) These changes do not impact mitochondrial morphology, as observed through (f) MitoTracker imaging (red) and (g) quantification in median fluorescence intensity (MFI) per mm 2 (Unt, n = 3; veh, n = 2; ZLN, n = 3). All gene expression data were analyzed using Student's t -test. Statistical significance is represented as follows: ns P > 0.05, ∗ P
    Mouse Anti Human α Tubulin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human α tubulin/product/Cell Signaling Technology Inc
    Average 99 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    mouse anti human α tubulin - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Millipore anti human α tubulin
    ZLN005 upregulates PGC-1 α and its downstream mitochondrial gene targets in ARPE-19 cells. (a) Relative gene expression (RE) of PGC-1α in ARPE-19 treated for 48 hours with 10 μ M ZLN005 (ZLN, n = 5) compared to vehicle (veh, n = 4). (b) Representative protein blot of PGC-1 α and <t>α</t> -tubulin bands and (c) quantification showed upregulation of PGC-1 α protein expression with ZLN005 treatment (all conditions, n = 4). (d) RPE-specific genes, BEST1 and RLBP1 , are upregulated with ZLN005 treatment (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (e) Treatment with 10 μ M ZLN005 increases the mitochondrial dynamic gene, MFN1 , and replication genes, TFAM and POLG (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (f, g) These changes do not impact mitochondrial morphology, as observed through (f) MitoTracker imaging (red) and (g) quantification in median fluorescence intensity (MFI) per mm 2 (Unt, n = 3; veh, n = 2; ZLN, n = 3). All gene expression data were analyzed using Student's t -test. Statistical significance is represented as follows: ns P > 0.05, ∗ P
    Anti Human α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human α tubulin/product/Millipore
    Average 99 stars, based on 45 article reviews
    Price from $9.99 to $1999.99
    anti human α tubulin - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    88
    Abcam rabbit anti human α tubulin
    ZLN005 upregulates PGC-1 α and its downstream mitochondrial gene targets in ARPE-19 cells. (a) Relative gene expression (RE) of PGC-1α in ARPE-19 treated for 48 hours with 10 μ M ZLN005 (ZLN, n = 5) compared to vehicle (veh, n = 4). (b) Representative protein blot of PGC-1 α and <t>α</t> -tubulin bands and (c) quantification showed upregulation of PGC-1 α protein expression with ZLN005 treatment (all conditions, n = 4). (d) RPE-specific genes, BEST1 and RLBP1 , are upregulated with ZLN005 treatment (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (e) Treatment with 10 μ M ZLN005 increases the mitochondrial dynamic gene, MFN1 , and replication genes, TFAM and POLG (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (f, g) These changes do not impact mitochondrial morphology, as observed through (f) MitoTracker imaging (red) and (g) quantification in median fluorescence intensity (MFI) per mm 2 (Unt, n = 3; veh, n = 2; ZLN, n = 3). All gene expression data were analyzed using Student's t -test. Statistical significance is represented as follows: ns P > 0.05, ∗ P
    Rabbit Anti Human α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti human α tubulin/product/Abcam
    Average 88 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    rabbit anti human α tubulin - by Bioz Stars, 2020-11
    88/100 stars
      Buy from Supplier

    99
    Abcam anti human α tubulin
    ZLN005 upregulates PGC-1 α and its downstream mitochondrial gene targets in ARPE-19 cells. (a) Relative gene expression (RE) of PGC-1α in ARPE-19 treated for 48 hours with 10 μ M ZLN005 (ZLN, n = 5) compared to vehicle (veh, n = 4). (b) Representative protein blot of PGC-1 α and <t>α</t> -tubulin bands and (c) quantification showed upregulation of PGC-1 α protein expression with ZLN005 treatment (all conditions, n = 4). (d) RPE-specific genes, BEST1 and RLBP1 , are upregulated with ZLN005 treatment (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (e) Treatment with 10 μ M ZLN005 increases the mitochondrial dynamic gene, MFN1 , and replication genes, TFAM and POLG (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (f, g) These changes do not impact mitochondrial morphology, as observed through (f) MitoTracker imaging (red) and (g) quantification in median fluorescence intensity (MFI) per mm 2 (Unt, n = 3; veh, n = 2; ZLN, n = 3). All gene expression data were analyzed using Student's t -test. Statistical significance is represented as follows: ns P > 0.05, ∗ P
    Anti Human α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human α tubulin/product/Abcam
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    anti human α tubulin - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    91
    Millipore α tubulin
    ZLN005 upregulates PGC-1 α and its downstream mitochondrial gene targets in ARPE-19 cells. (a) Relative gene expression (RE) of PGC-1α in ARPE-19 treated for 48 hours with 10 μ M ZLN005 (ZLN, n = 5) compared to vehicle (veh, n = 4). (b) Representative protein blot of PGC-1 α and <t>α</t> -tubulin bands and (c) quantification showed upregulation of PGC-1 α protein expression with ZLN005 treatment (all conditions, n = 4). (d) RPE-specific genes, BEST1 and RLBP1 , are upregulated with ZLN005 treatment (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (e) Treatment with 10 μ M ZLN005 increases the mitochondrial dynamic gene, MFN1 , and replication genes, TFAM and POLG (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (f, g) These changes do not impact mitochondrial morphology, as observed through (f) MitoTracker imaging (red) and (g) quantification in median fluorescence intensity (MFI) per mm 2 (Unt, n = 3; veh, n = 2; ZLN, n = 3). All gene expression data were analyzed using Student's t -test. Statistical significance is represented as follows: ns P > 0.05, ∗ P
    α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 21797 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α tubulin/product/Millipore
    Average 91 stars, based on 21797 article reviews
    Price from $9.99 to $1999.99
    α tubulin - by Bioz Stars, 2020-11
    91/100 stars
      Buy from Supplier

    99
    Abcam human α tubulin
    SmCBP1 and SmGCN5 play a key role in Smp14 expression and female sexual reproductive development. A dsRNAi experiment was carried out on sixteen adult worm pairs cultivated for seven days. Worms were electroporated and soaked with dsRNAi from SmCBP1 or SmGCN5 or with the non-specific dsRNAi LUC. On the seventh day of culture, the mRNA levels of SmCBP1, SmGCN5 or Smp14 (graphs A and B) were determined by qRT-PCR, normalized by the <t>α-tubulin</t> transcript levels. The results are depicted in relation to the non-specific dsRNAi (dsLUC) mRNA levels. The effects of the dsRNAi on targeted-protein synthesis were assessed by Western blot (panels C and D). The total number of eggs laid by the same parasites that received the dsRNAi was counted (graphs E and F). Adult worms from the dsRNAi experiments were analyzed by confocal laser scanning microscopy (G). Worms received either the control dsRNAi LUC (panels a and d) or the dsRNAi for SmCBP1 (panels b and e) or SmGCN5 (panels c and f). Panels a–c, and d–f, reveal the effect of the dsRNAi in the ovary, and vitellaria, respectively. OV: ovary; mo: mature oocytes; io: immature oocytes; v: vitellaria; rs: receptaculum seminis. Results are pooled from three independent experiments. Western blots were repeated three times. The confocal microscopy images are representative of several parasites analyzed. Scale bars 10 µm. Student's t-test was applied, with *p
    Human α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human α tubulin/product/Abcam
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    human α tubulin - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    91
    Abcam rabbit polyclonal anti human α tubulin
    MTs are twisting around IFs in TnT as observed with confocal microscopy. ( a ) Positions 1–5 correspond to ( b ) x-z and y-z projections and show that MTs <t>(α-tubulin,</t> green) rotate around IF core (CK7, red). Note the rotation of MTs (arrowheads) in x-z and in y-z. ( c ) Gray values of MTs (α-tubulin) and IFs (CK7) fluorescence intensity along TnT are plotted. Gray values of α-tubulin reach point 0 between two peaks, which correspond to one twist of MTs around IFs. Presented normal urothelial cells were methanol fixed.
    Rabbit Polyclonal Anti Human α Tubulin, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti human α tubulin/product/Abcam
    Average 91 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti human α tubulin - by Bioz Stars, 2020-11
    91/100 stars
      Buy from Supplier

    99
    Millipore mouse anti human α tubulin
    Ars2 is commonly expressed in glioblastoma cells. (A) Real-time qPCR analysis of Ars2 mRNA expression levels in four glioblastoma cell lines (U87, A172, LN-229 and U251) and one normal astrocyte cell line (HEB). The data represent the average obtained from three independent experiments and are presented as the mean ± SD. (B) Western blot analysis of Ars2 expression in four glioblastoma cell lines and one normal astrocyte cell line. <t>α-tubulin</t> is shown as the loading control. (C) Immunohistochemical analysis of Ars2 expression in human glioblastoma tumor and paired normal tissues. Scale bar, 10 µm. (D) Statistical analyses of immunohistochemical results of Ars2 expression levels in 12 paired samples of normal and glioblastoma tissues; P=0.0001. Ars2, arsenic resistance protein 2.
    Mouse Anti Human α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human α tubulin/product/Millipore
    Average 99 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
    mouse anti human α tubulin - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    91
    Thermo Fisher mouse anti human α tubulin
    PDAC microtumours are triggered by a cell-in-cell structure anchored to a microisland and are covered by membranous expression of <t>α-tubulin.</t> ( a ). ( b ). ( c ) When a mix of CFSE-labelled PCI-55 cells and unlabelled PCI-55 cells were sparsely cultured, they showed a cell-in-cell structure on the rough circular microisland. ( d ) Papillary PCI-55 microtumour formed by cell-in-cell structures. ( e , f ) Immunofluorescent staining for α-tubulin in the microtumours self-organised by CFSE-labelled PCI-55 cells. CFSE High -labelled PCI-55 cells penetrating CFSE Low -labelled cells that formed a microtumour ( e ). Multinucleated giant PCI-55 cells containing at least six high CFSE-labelled PCI-55 cells ( f , g ). The surface of a papillary microtumour self-organised by PCI-55 cells. Bars: 30 µm. ( h ) Many PCI-55 cells in a mitotic phase were outside the surface of the microtumours. Z-stack full-focus images of immunofluorescent staining for α-tubulin in PCI-55 microtumours anchored to the micro/nanoplate. ( i ) Counts of mitotic cells in PCI-55 microtumours. ( j , k ) ( j ). 3D images ( k ). ( l ). m, Immunofluorescent staining for α-tubulin in PCI-55 cells colonised on the peritoneum. CFSE-labelled PCI-55 cells were grafted intraperitoneally into SCID mice. After 3 days, the peritonea were extracted.
    Mouse Anti Human α Tubulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti human α tubulin/product/Thermo Fisher
    Average 91 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    mouse anti human α tubulin - by Bioz Stars, 2020-11
    91/100 stars
      Buy from Supplier

    Image Search Results


    Histochemistry analysis of brain sections from patient AD1. Evidence of CA in the brain cortex containing microbial proteins. Insula cortex (INCO) sections from patient AD1 stained with Periodic acid–Schiff reagent (upper panel); immunostained with a rabbit polyclonal anti- C. albicans antibody (green) used at 1:100 dilution, and a mouse monoclonal anti-human α-tubulin antibody (red) used at 1:50 dilution (middle panel); immunostained with a mouse monoclonal anti-peptidoglycan antibody (green) used at 1:20 dilution, and a rabbit polyclonal anti- C . albicans antibody (red) used at 1:100 dilution (lower panel). DAPI staining of nuclei appears in blue. Scale bar: 50 μm.

    Journal: Scientific Reports

    Article Title: Human and Microbial Proteins From Corpora Amylacea of Alzheimer’s Disease

    doi: 10.1038/s41598-018-28231-1

    Figure Lengend Snippet: Histochemistry analysis of brain sections from patient AD1. Evidence of CA in the brain cortex containing microbial proteins. Insula cortex (INCO) sections from patient AD1 stained with Periodic acid–Schiff reagent (upper panel); immunostained with a rabbit polyclonal anti- C. albicans antibody (green) used at 1:100 dilution, and a mouse monoclonal anti-human α-tubulin antibody (red) used at 1:50 dilution (middle panel); immunostained with a mouse monoclonal anti-peptidoglycan antibody (green) used at 1:20 dilution, and a rabbit polyclonal anti- C . albicans antibody (red) used at 1:100 dilution (lower panel). DAPI staining of nuclei appears in blue. Scale bar: 50 μm.

    Article Snippet: Mouse monoclonal antibodies against peptidoglycan and human α-tubulin were purchased from Sigma and Thermo Scientific, respectively.

    Techniques: Staining

    Effects of AICAR on mitochondrial apoptosis in human osteosarcoma xenografts. (A) DNA fragmentation in KHOS cell-derived tumor tissues after 14 days of AICAR treatment was assessed by flow cytometry. (B and C) Immunoblot analysis of (B) apoptosis-related proteins and (C) mitochondrial factors (PGC-1α and TFAM) in AICAR-treated or control KHOS cell-derived tumor tissues. Positive bands were semiquantified by densitometrical analyses using the ImageJ software. Values were normalized against α-tubulin and presented as a ratio. (D and E) Mitochondrial proliferation and DNA fragmentation in (D) MG63 and (E) KHOS cell-derived tumor tissues from AICAR-treated and control mice were evaluated by immunofluorescence staining. Red, mitochondria (MitoTracker Deep-Red FM); green, apoptotic nuclei (APO-Direct); blue, nuclei (DAPI).

    Journal: International Journal of Oncology

    Article Title: AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway

    doi: 10.3892/ijo.2016.3775

    Figure Lengend Snippet: Effects of AICAR on mitochondrial apoptosis in human osteosarcoma xenografts. (A) DNA fragmentation in KHOS cell-derived tumor tissues after 14 days of AICAR treatment was assessed by flow cytometry. (B and C) Immunoblot analysis of (B) apoptosis-related proteins and (C) mitochondrial factors (PGC-1α and TFAM) in AICAR-treated or control KHOS cell-derived tumor tissues. Positive bands were semiquantified by densitometrical analyses using the ImageJ software. Values were normalized against α-tubulin and presented as a ratio. (D and E) Mitochondrial proliferation and DNA fragmentation in (D) MG63 and (E) KHOS cell-derived tumor tissues from AICAR-treated and control mice were evaluated by immunofluorescence staining. Red, mitochondria (MitoTracker Deep-Red FM); green, apoptotic nuclei (APO-Direct); blue, nuclei (DAPI).

    Article Snippet: Membranes were reprobed with anti-human α-tubulin antibody (Sigma-Aldrich) to confirm equal protein loading.

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Pyrolysis Gas Chromatography, Software, Mouse Assay, Immunofluorescence, Staining

    AMPK phosphorylation and mitochondrial proliferation after AICAR treatment in osteosarcoma cells. (A and B) The levels of AMPKα and phosphorylated AMPKα (Thr172) were evaluated by immunoblot analyses after 30 min, 1 and 6 h of AICAR treatment (1,000 µ M) in (A) MG63 and (B) KHOS osteosarcoma cells. Positive bands were semiquantified by densitometrical analyses using ImageJ software. Values were normalized against α-tubulin and presented as a ratio. (C) Relative mtDNA copy numbers to nDNA in AICAR-treated osteosarcoma cell lines were evaluated by quantitative real-time PCR after 72 h of treatment. Data represent the mean ± SEM of at least three independent experiments ( * p

    Journal: International Journal of Oncology

    Article Title: AICAR induces mitochondrial apoptosis in human osteosarcoma cells through an AMPK-dependent pathway

    doi: 10.3892/ijo.2016.3775

    Figure Lengend Snippet: AMPK phosphorylation and mitochondrial proliferation after AICAR treatment in osteosarcoma cells. (A and B) The levels of AMPKα and phosphorylated AMPKα (Thr172) were evaluated by immunoblot analyses after 30 min, 1 and 6 h of AICAR treatment (1,000 µ M) in (A) MG63 and (B) KHOS osteosarcoma cells. Positive bands were semiquantified by densitometrical analyses using ImageJ software. Values were normalized against α-tubulin and presented as a ratio. (C) Relative mtDNA copy numbers to nDNA in AICAR-treated osteosarcoma cell lines were evaluated by quantitative real-time PCR after 72 h of treatment. Data represent the mean ± SEM of at least three independent experiments ( * p

    Article Snippet: Membranes were reprobed with anti-human α-tubulin antibody (Sigma-Aldrich) to confirm equal protein loading.

    Techniques: Software, Real-time Polymerase Chain Reaction

    Inflammation induces EndoMT in BECs. a Confluent BECs were stimulated with TGF-β1 and IL-1β as described in the materials and method section, mRNA expression levels of different EndoMT-associated TFs, and endothelial or mesenchymal markers were determined by qRT-PCR. Values were normalized to GAPDH and plotted as fold change relative to control (dotted line). b – f TGF-β1- and IL-1β-stimulated BECs total and nuclear protein levels of SNAI1, CLDN5, CDH5, and CDH2 was assessed by western blot and expressed as fold change compared to control cells. Values were normalized to β-actin, α-tubulin or Lamin B levels. g – p CLDN5, CDH5, OCLN, CDH2, and FSP1 protein expression was assessed by immunofluorescence and imaged under the confocal microscope (scale bar 20 µm). Data presented are the mean of triplicate values ± SEM of three independent experiments. Statistical analysis was performed using Student’s t -test or one-way ANOVA, where * p

    Journal: Cell Death & Disease

    Article Title: Inflammation-induced endothelial to mesenchymal transition promotes brain endothelial cell dysfunction and occurs during multiple sclerosis pathophysiology

    doi: 10.1038/s41419-018-1294-2

    Figure Lengend Snippet: Inflammation induces EndoMT in BECs. a Confluent BECs were stimulated with TGF-β1 and IL-1β as described in the materials and method section, mRNA expression levels of different EndoMT-associated TFs, and endothelial or mesenchymal markers were determined by qRT-PCR. Values were normalized to GAPDH and plotted as fold change relative to control (dotted line). b – f TGF-β1- and IL-1β-stimulated BECs total and nuclear protein levels of SNAI1, CLDN5, CDH5, and CDH2 was assessed by western blot and expressed as fold change compared to control cells. Values were normalized to β-actin, α-tubulin or Lamin B levels. g – p CLDN5, CDH5, OCLN, CDH2, and FSP1 protein expression was assessed by immunofluorescence and imaged under the confocal microscope (scale bar 20 µm). Data presented are the mean of triplicate values ± SEM of three independent experiments. Statistical analysis was performed using Student’s t -test or one-way ANOVA, where * p

    Article Snippet: Subsequently, blots were incubated in blocking buffer containing 0.1% Tween-20 with antibodies against claudin-5 (CLDN5) (Thermo Fisher Scientific, Rockford, IL, USA), VE-cadherin (CDH5) (BD Biosciences, Franklin Lakes, NJ, USA), CDH2 (Sigma-Aldrich, Saint Louis, MO, USA), SNAI1 (Thermo Fisher Scientific, Rockford, IL, USA), β-actin (Santa Cruz, Dallas, TX, USA), α-tubulin (Clone DM1A, Cedarlane Laboratories, Burlington, Canada) and Lamin B (Santa Cruz, Dallas, TX, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Microscopy

    EndoMT can be reverted by inhibiting TAK1. a Confluent BECs were stimulated as indicated in the figure and as described in the materials and method section. Protein levels of phospho-TAK1 (Thr184/187) in cell lysates were determined by Western blot. Values were normalized to α-tubulin and plotted as fold change relative to control. b – e Stimulated-BECs were stained for NF-kB (p65 subunit) and analyzed using the A1R+ confocal resonant scanning laser microscope from Nikon (scale bar 20 µm). f NF-kB (p65 subunit) nuclear staining intensity was quantified using NIN ImageJ software analysis in three random fields in each well and is expressed as fold change intensity relative to unstimulated control cells. g mRNA expression levels of different EndoMT-related markers were determined by qRT-PCR upon stimulation of BECs. Values were normalized to GAPDH and plotted as fold change relative to control. h Nuclear protein content of SNAI1 was assessed by western blot and expressed as fold change compared to control cells. SnaI1 nuclear protein content was normalized using the nuclear envelope protein Lamin B. i , j The TEER (Rb, barrier resistance) and permeability to FITC-dextran (A.U. arbitrary unit) were measured and plotted as % of control BECs. Data presented are the mean of triplicate values ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA where * p

    Journal: Cell Death & Disease

    Article Title: Inflammation-induced endothelial to mesenchymal transition promotes brain endothelial cell dysfunction and occurs during multiple sclerosis pathophysiology

    doi: 10.1038/s41419-018-1294-2

    Figure Lengend Snippet: EndoMT can be reverted by inhibiting TAK1. a Confluent BECs were stimulated as indicated in the figure and as described in the materials and method section. Protein levels of phospho-TAK1 (Thr184/187) in cell lysates were determined by Western blot. Values were normalized to α-tubulin and plotted as fold change relative to control. b – e Stimulated-BECs were stained for NF-kB (p65 subunit) and analyzed using the A1R+ confocal resonant scanning laser microscope from Nikon (scale bar 20 µm). f NF-kB (p65 subunit) nuclear staining intensity was quantified using NIN ImageJ software analysis in three random fields in each well and is expressed as fold change intensity relative to unstimulated control cells. g mRNA expression levels of different EndoMT-related markers were determined by qRT-PCR upon stimulation of BECs. Values were normalized to GAPDH and plotted as fold change relative to control. h Nuclear protein content of SNAI1 was assessed by western blot and expressed as fold change compared to control cells. SnaI1 nuclear protein content was normalized using the nuclear envelope protein Lamin B. i , j The TEER (Rb, barrier resistance) and permeability to FITC-dextran (A.U. arbitrary unit) were measured and plotted as % of control BECs. Data presented are the mean of triplicate values ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA where * p

    Article Snippet: Subsequently, blots were incubated in blocking buffer containing 0.1% Tween-20 with antibodies against claudin-5 (CLDN5) (Thermo Fisher Scientific, Rockford, IL, USA), VE-cadherin (CDH5) (BD Biosciences, Franklin Lakes, NJ, USA), CDH2 (Sigma-Aldrich, Saint Louis, MO, USA), SNAI1 (Thermo Fisher Scientific, Rockford, IL, USA), β-actin (Santa Cruz, Dallas, TX, USA), α-tubulin (Clone DM1A, Cedarlane Laboratories, Burlington, Canada) and Lamin B (Santa Cruz, Dallas, TX, USA).

    Techniques: Western Blot, Staining, Microscopy, Software, Expressing, Quantitative RT-PCR, Permeability

    Increased hepatic IP-10 expression and liver homing of NK cells following iNKT cell activation. Livers of wt mice were excised 12 h after s.c. administration of a single dose of αGalCerMPEG (10 µg) and prepared for histopathological or Western blot analysis. ( A – F ) histopathological analysis of IP-10 expression (brown) in the liver of untreated ( A – C ) and treated ( D – F ) mice at an original magnification of x10 ( A , D ) and x20 ( B , C and E , F ). ( G ) Western blot analysis of monomeric IP-10 expression in hepatocytes isolated from untreated and αGalCerMPEG- administered mice (n = 9–11 mice, shown is one out of at least three independent experiments). The data are presented as the ratio of the volume of integrals of the signals detected for IP-10 and α-tubulin. For the in vivo migration studies, CD45.1 + NK cells were adoptively transferred intravenously (i.v.) into wt mice. Recipient mice were administered s.c. a single dose of αGalCerMPEG (10 µg) 24 h later and lymphocytes derived from spleen and liver were analyzed 72 h after treatment. Frequencies and absolute numbers of CD3 − NK1.1 + CD45.1 + cells (out of 1 × 10 6 total cells) derived from ( H ) the spleen and ( I ) the liver of untreated (white) and treated (black) mice. Shown are the compiled data derived from two independent experiments (n = 7–8 mice). ( J ) The mechanism of NK cell migration was assessed using a trans-well system. Hepatocytes, serum and NK cells were obtained from untreated (−) or treated (+) mice 12 h after administration of a single dose of αGalCerMPEG (s.c., 10 µg). Absolute numbers of CFSE + NK cells (out of 1 × 10 6 total cells) were assessed after 2 h incubation at 37 °C (n = 3 mice, shown is one out of two independent experiments). Columns represent the mean ± SEM and circles indicate single values. Violin plots represent the interquartile range, horizontal lines show the mean value and the width displays the distribution of data points. Asterisks denote significant values as calculated by unpaired, two-tailed Student’s t-test as compared to untreated control. **p ≤ 0.01, *p ≤ 0.05, n.s. = not significant.

    Journal: Scientific Reports

    Article Title: Key features and homing properties of NK cells in the liver are shaped by activated iNKT cells

    doi: 10.1038/s41598-019-52666-9

    Figure Lengend Snippet: Increased hepatic IP-10 expression and liver homing of NK cells following iNKT cell activation. Livers of wt mice were excised 12 h after s.c. administration of a single dose of αGalCerMPEG (10 µg) and prepared for histopathological or Western blot analysis. ( A – F ) histopathological analysis of IP-10 expression (brown) in the liver of untreated ( A – C ) and treated ( D – F ) mice at an original magnification of x10 ( A , D ) and x20 ( B , C and E , F ). ( G ) Western blot analysis of monomeric IP-10 expression in hepatocytes isolated from untreated and αGalCerMPEG- administered mice (n = 9–11 mice, shown is one out of at least three independent experiments). The data are presented as the ratio of the volume of integrals of the signals detected for IP-10 and α-tubulin. For the in vivo migration studies, CD45.1 + NK cells were adoptively transferred intravenously (i.v.) into wt mice. Recipient mice were administered s.c. a single dose of αGalCerMPEG (10 µg) 24 h later and lymphocytes derived from spleen and liver were analyzed 72 h after treatment. Frequencies and absolute numbers of CD3 − NK1.1 + CD45.1 + cells (out of 1 × 10 6 total cells) derived from ( H ) the spleen and ( I ) the liver of untreated (white) and treated (black) mice. Shown are the compiled data derived from two independent experiments (n = 7–8 mice). ( J ) The mechanism of NK cell migration was assessed using a trans-well system. Hepatocytes, serum and NK cells were obtained from untreated (−) or treated (+) mice 12 h after administration of a single dose of αGalCerMPEG (s.c., 10 µg). Absolute numbers of CFSE + NK cells (out of 1 × 10 6 total cells) were assessed after 2 h incubation at 37 °C (n = 3 mice, shown is one out of two independent experiments). Columns represent the mean ± SEM and circles indicate single values. Violin plots represent the interquartile range, horizontal lines show the mean value and the width displays the distribution of data points. Asterisks denote significant values as calculated by unpaired, two-tailed Student’s t-test as compared to untreated control. **p ≤ 0.01, *p ≤ 0.05, n.s. = not significant.

    Article Snippet: Membranes were incubated first with the primary antibodies against IP-10 or α-tubulin (goat anti mouse IP-10, R & D Systems; rabbit anti mouse α-tubulin, Abcam) at 4 °C overnight and second with the respective horseradish-peroxidase-conjugated antibodies.

    Techniques: Expressing, Activation Assay, Mouse Assay, Western Blot, Isolation, In Vivo, Migration, Derivative Assay, Incubation, Two Tailed Test

    The expression of CD74 and CD44 receptors in immortalized normal breast luminal cells (226LDM) (A) Cell-surface and intracellular expression of CD74 and CD44 was acquired by flow cytometry using By2 (anti-CD74) and 156-3C11 (anti-CD44). Black-filled histograms represent the 226LDM cells stained with indicated antibody. Empty histograms show the isotype as negative controls. (B) Total protein of CD74 and CD44 was detected by Western blotting, and α-tubulin was used as a loading control. (C) Confocal images of CD74 and CD44 in 226LDM cells stained intracellularly: CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). Figures depict representative samples from duplicate experiments. Scale bar 10 μm.

    Journal: Oncotarget

    Article Title: Measurements of heterotypic associations between cluster of differentiation CD74 and CD44 in human breast cancer-derived cells

    doi: 10.18632/oncotarget.20922

    Figure Lengend Snippet: The expression of CD74 and CD44 receptors in immortalized normal breast luminal cells (226LDM) (A) Cell-surface and intracellular expression of CD74 and CD44 was acquired by flow cytometry using By2 (anti-CD74) and 156-3C11 (anti-CD44). Black-filled histograms represent the 226LDM cells stained with indicated antibody. Empty histograms show the isotype as negative controls. (B) Total protein of CD74 and CD44 was detected by Western blotting, and α-tubulin was used as a loading control. (C) Confocal images of CD74 and CD44 in 226LDM cells stained intracellularly: CD74 was labelled with Alexa Fluor 488 (green) and CD44 with Alexa Fluor 555 (red). Figures depict representative samples from duplicate experiments. Scale bar 10 μm.

    Article Snippet: Reagents The monoclonal primary antibodies mouse anti-human CD74 (clone: By2) and mouse anti-human α-tubulin (clone: TU-02) were purchased from Santa Cruz Biotechnology, USA.

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Western Blot

    MUC16 protein expression in cultured cells after MUC16 gene editing ( A–C ) Western blot analysis of MUC16 protein levels in the three cell lines after transfection alone (Transfection), transfection plus short-term cisplatin treatment (Short cis treatment), and transfection plus long-term cisplatin treatment (Long cis treatment). Compared to those of the parent cell lines (P and Parent), elevated protein levels were observed in cultured cells after MUC16 gene editing. The MUC16 protein band corresponding to 180 kDa (left) was used for semi-quantitative analysis. α-Tubulin (50 kDa) was used as the internal control and reference for semi-quantitative analysis. ( D ) MUC16 protein was stained in the three cell lines using immunofluorescent antibodies. The staining signal was slightly increased at the membranes and in the cytoplasm of A549 and 293T cells after treatment compared to that of the parent cells. Note: the staining was obviously enhanced at the membranes of EPLC-32M1 cells after treatment. Parent: parent cells (wild types); Trans: transfection alone; Cis: transfection plus long-term cisplatin treatment. S1, S2, S2-1, S4, S5, S5-1, and R1: mutation systems used in this study.

    Journal: Oncotarget

    Article Title: MUC16 overexpression induced by gene mutations promotes lung cancer cell growth and invasion

    doi: 10.18632/oncotarget.24203

    Figure Lengend Snippet: MUC16 protein expression in cultured cells after MUC16 gene editing ( A–C ) Western blot analysis of MUC16 protein levels in the three cell lines after transfection alone (Transfection), transfection plus short-term cisplatin treatment (Short cis treatment), and transfection plus long-term cisplatin treatment (Long cis treatment). Compared to those of the parent cell lines (P and Parent), elevated protein levels were observed in cultured cells after MUC16 gene editing. The MUC16 protein band corresponding to 180 kDa (left) was used for semi-quantitative analysis. α-Tubulin (50 kDa) was used as the internal control and reference for semi-quantitative analysis. ( D ) MUC16 protein was stained in the three cell lines using immunofluorescent antibodies. The staining signal was slightly increased at the membranes and in the cytoplasm of A549 and 293T cells after treatment compared to that of the parent cells. Note: the staining was obviously enhanced at the membranes of EPLC-32M1 cells after treatment. Parent: parent cells (wild types); Trans: transfection alone; Cis: transfection plus long-term cisplatin treatment. S1, S2, S2-1, S4, S5, S5-1, and R1: mutation systems used in this study.

    Article Snippet: Mouse anti-human MUC16 monoclonal antibody (mAb) (stock: 764 µg/ml dilution factor 1:1000) and mouse anti-human α-tubulin mAb (diluted 1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were the primary antibodies used.

    Techniques: Expressing, Cell Culture, Western Blot, Transfection, Staining, Mutagenesis

    ERK activation mediates UVB-induced TM down-regulation in HaCaT cells. (A) HaCaT cells were pre-treated with 10 µM pharmacological inhibitors followed by UVB irradiation (3 mJ/cm 2 ) for 24 h. TM protein levels were analyzed by Western blotting. α-tubulin served as an internal control. (B) HaCaT cells preincubated with 10 µM U0126 or SB203580 were irradiated with UVB (3 mJ/cm 2 ). TM, pERK, and p-p38 protein levels were determined by Western blotting. α-tubulin, ERK, or p38 levels are used as loading controls, respectively. (C) Intracellular ROS levels were measured with 10 µM DCF-DA 30 min after UVB irradiation. The results are expressed as the mean ± S.D. of three independent experiments. (D) HaCaT cells were pretreated for 1 h with NAC followed by UVB irradiation (3 mJ/cm 2 ) for 24 h, and TM and pERK protein levels were examined by Western blotting. α-tubulin and total ERK were used as loading controls. Relative TM or ERK phosphorylation levels were quantified and presented as the mean ± SD for three independent experiments performed in triplicate. #, p

    Journal: PLoS ONE

    Article Title: UVB Irradiation Regulates ERK1/2- and p53-Dependent Thrombomodulin Expression in Human Keratinocytes

    doi: 10.1371/journal.pone.0067632

    Figure Lengend Snippet: ERK activation mediates UVB-induced TM down-regulation in HaCaT cells. (A) HaCaT cells were pre-treated with 10 µM pharmacological inhibitors followed by UVB irradiation (3 mJ/cm 2 ) for 24 h. TM protein levels were analyzed by Western blotting. α-tubulin served as an internal control. (B) HaCaT cells preincubated with 10 µM U0126 or SB203580 were irradiated with UVB (3 mJ/cm 2 ). TM, pERK, and p-p38 protein levels were determined by Western blotting. α-tubulin, ERK, or p38 levels are used as loading controls, respectively. (C) Intracellular ROS levels were measured with 10 µM DCF-DA 30 min after UVB irradiation. The results are expressed as the mean ± S.D. of three independent experiments. (D) HaCaT cells were pretreated for 1 h with NAC followed by UVB irradiation (3 mJ/cm 2 ) for 24 h, and TM and pERK protein levels were examined by Western blotting. α-tubulin and total ERK were used as loading controls. Relative TM or ERK phosphorylation levels were quantified and presented as the mean ± SD for three independent experiments performed in triplicate. #, p

    Article Snippet: The membrane was blocked in 5% fat-free milk in PBST buffer (PBS with 0.05% Tween-20) followed by incubation overnight with primary antibodies (mouse anti-human TM antibodies (1∶1000), mouse anti-human pERK antibodies (1∶1000), mouse anti-human ERK antibodies (1∶1000), mouse anti-human α-tubulin antibodies (1∶2000), mouse anti-human GAPDH antibodies (1∶2000) (Santa Cruz Biotech, CA, USA), mouse anti-human p53 antibodies (1∶1000), mouse anti-human NF-κB antibodies (1∶1000), mouse anti-human fibrillarin antibodies (1∶2000), and mouse anti-human lamin B1 antibody (1∶1000) (Abcam, Cambridge, MA) diluted in PBST buffer.

    Techniques: Activation Assay, Irradiation, Western Blot

    p53 mediated UVB-induced TM downregulation. (A) HaCaT cells (upper panel) or primary human epidermal keratinocytes (lower panel) were UVB irradiated (3 mJ/cm 2 ) at the indicated time points, and cytosolic and nuclear fractions were harvested. p53 and p65 protein levels were determined by Western blotting. α-tubulin or lamin B1 levels were used as cytoplasmic or nuclear loading controls, respectively. (B) Nuclear extracts from UVB-irradiated HaCaT cells (left panel) or human epidermal keratinocytes (right panel) were incubated with proximal TM promoter DNA linked to Dynabeads. Protein-DNA complexes were allowed to form under native conditions and precipitated magnetically. Bound proteins were washed, eluted, and p53 and p65 levels were determined by Western blotting (C) HaCaT cells were UVB irradiated (3 mJ/cm 2 ) for 3 h, and chromatin immunoprecipitation was performed. Chromatin was immunoprecipitated with anti-p53 or anti-p65 antibodies followed by amplification of TM promoter elements by PCR using specific primers. To verify equal loading, 1% of the precipitated chromatin was assayed as input. (D) HaCaT cells were transiently transfected with p53 siRNA or with a GAPDH short hairpin RNA (shRNA) as control. After transfection, cells were exposed to UVB (3 mJ/cm 2 ) for 24 h, and cell lysate was harvested for Western blot analysis to assess p53 and TM levels. α-tubulin was used as a loading control.

    Journal: PLoS ONE

    Article Title: UVB Irradiation Regulates ERK1/2- and p53-Dependent Thrombomodulin Expression in Human Keratinocytes

    doi: 10.1371/journal.pone.0067632

    Figure Lengend Snippet: p53 mediated UVB-induced TM downregulation. (A) HaCaT cells (upper panel) or primary human epidermal keratinocytes (lower panel) were UVB irradiated (3 mJ/cm 2 ) at the indicated time points, and cytosolic and nuclear fractions were harvested. p53 and p65 protein levels were determined by Western blotting. α-tubulin or lamin B1 levels were used as cytoplasmic or nuclear loading controls, respectively. (B) Nuclear extracts from UVB-irradiated HaCaT cells (left panel) or human epidermal keratinocytes (right panel) were incubated with proximal TM promoter DNA linked to Dynabeads. Protein-DNA complexes were allowed to form under native conditions and precipitated magnetically. Bound proteins were washed, eluted, and p53 and p65 levels were determined by Western blotting (C) HaCaT cells were UVB irradiated (3 mJ/cm 2 ) for 3 h, and chromatin immunoprecipitation was performed. Chromatin was immunoprecipitated with anti-p53 or anti-p65 antibodies followed by amplification of TM promoter elements by PCR using specific primers. To verify equal loading, 1% of the precipitated chromatin was assayed as input. (D) HaCaT cells were transiently transfected with p53 siRNA or with a GAPDH short hairpin RNA (shRNA) as control. After transfection, cells were exposed to UVB (3 mJ/cm 2 ) for 24 h, and cell lysate was harvested for Western blot analysis to assess p53 and TM levels. α-tubulin was used as a loading control.

    Article Snippet: The membrane was blocked in 5% fat-free milk in PBST buffer (PBS with 0.05% Tween-20) followed by incubation overnight with primary antibodies (mouse anti-human TM antibodies (1∶1000), mouse anti-human pERK antibodies (1∶1000), mouse anti-human ERK antibodies (1∶1000), mouse anti-human α-tubulin antibodies (1∶2000), mouse anti-human GAPDH antibodies (1∶2000) (Santa Cruz Biotech, CA, USA), mouse anti-human p53 antibodies (1∶1000), mouse anti-human NF-κB antibodies (1∶1000), mouse anti-human fibrillarin antibodies (1∶2000), and mouse anti-human lamin B1 antibody (1∶1000) (Abcam, Cambridge, MA) diluted in PBST buffer.

    Techniques: Irradiation, Western Blot, Incubation, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Transfection, shRNA

    UVB irradiation decreased TM expression in HaCaT cells. (A) HaCaT Cells were exposed to UVB irradiation in a dose-response (0, 1.5, 3, 4.5, 6 mJ/cm 2 ) Cell viability was examined by trypan blue exclusion. (B) HaCaT cells were exposed to 1.5 or 3 mJ/cm 2 UVB for 24 h, and TM activity was determined by protein C activation analysis and expressed as fold change from the non-irradiated control. (C) (Upper panels) Western blot results of the UVB irradiation-mediated decrease in TM protein levels. α-tubulin served as an internal control. HaCaT cells were irradiated with 1.5, or 3 mJ/cm2 of UVB for 12, 24 or 48 h or without irradiation, respectively. On the other hand, primary human keratinocytes were irradiated with 3 mJ/cm2 of UVB for 24 or 48 h or without UVB treatment. (Lower panel) Quantification of Western blots from (C). The untreated cells at each time point served as a control for each time point. The results are presented as the mean ± standard deviation from three independent experiments. (D) Total RNA from UVB-irradiated HaCaT cells at the indicated doses was collected. TM mRNA levels were examined by real time RT-PCR using GAPDH as an internal control. (* p

    Journal: PLoS ONE

    Article Title: UVB Irradiation Regulates ERK1/2- and p53-Dependent Thrombomodulin Expression in Human Keratinocytes

    doi: 10.1371/journal.pone.0067632

    Figure Lengend Snippet: UVB irradiation decreased TM expression in HaCaT cells. (A) HaCaT Cells were exposed to UVB irradiation in a dose-response (0, 1.5, 3, 4.5, 6 mJ/cm 2 ) Cell viability was examined by trypan blue exclusion. (B) HaCaT cells were exposed to 1.5 or 3 mJ/cm 2 UVB for 24 h, and TM activity was determined by protein C activation analysis and expressed as fold change from the non-irradiated control. (C) (Upper panels) Western blot results of the UVB irradiation-mediated decrease in TM protein levels. α-tubulin served as an internal control. HaCaT cells were irradiated with 1.5, or 3 mJ/cm2 of UVB for 12, 24 or 48 h or without irradiation, respectively. On the other hand, primary human keratinocytes were irradiated with 3 mJ/cm2 of UVB for 24 or 48 h or without UVB treatment. (Lower panel) Quantification of Western blots from (C). The untreated cells at each time point served as a control for each time point. The results are presented as the mean ± standard deviation from three independent experiments. (D) Total RNA from UVB-irradiated HaCaT cells at the indicated doses was collected. TM mRNA levels were examined by real time RT-PCR using GAPDH as an internal control. (* p

    Article Snippet: The membrane was blocked in 5% fat-free milk in PBST buffer (PBS with 0.05% Tween-20) followed by incubation overnight with primary antibodies (mouse anti-human TM antibodies (1∶1000), mouse anti-human pERK antibodies (1∶1000), mouse anti-human ERK antibodies (1∶1000), mouse anti-human α-tubulin antibodies (1∶2000), mouse anti-human GAPDH antibodies (1∶2000) (Santa Cruz Biotech, CA, USA), mouse anti-human p53 antibodies (1∶1000), mouse anti-human NF-κB antibodies (1∶1000), mouse anti-human fibrillarin antibodies (1∶2000), and mouse anti-human lamin B1 antibody (1∶1000) (Abcam, Cambridge, MA) diluted in PBST buffer.

    Techniques: Irradiation, Expressing, Activity Assay, Activation Assay, Western Blot, Standard Deviation, Quantitative RT-PCR

    C14orf166 upregulation in bladder cancer tissues. a Quantitative RT-PCR determination of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues from six patients. Transcription levels were normalized to GAPDH expression. b Western blot analysis of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues. α-Tubulin was used as the loading control. c IHC determination of C14orf166 expression in different patients with different clinical stages of disease according to T classification (magnification: ×200, ×400)

    Journal: Journal of Translational Medicine

    Article Title: C14orf166 is a high-risk biomarker for bladder cancer and promotes bladder cancer cell proliferation

    doi: 10.1186/s12967-016-0801-4

    Figure Lengend Snippet: C14orf166 upregulation in bladder cancer tissues. a Quantitative RT-PCR determination of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues from six patients. Transcription levels were normalized to GAPDH expression. b Western blot analysis of C14orf166 expression in bladder cancer tissues and adjacent bladder tissues. α-Tubulin was used as the loading control. c IHC determination of C14orf166 expression in different patients with different clinical stages of disease according to T classification (magnification: ×200, ×400)

    Article Snippet: An antibody to human α-Tubulin (1:5000, Santa Cruz) was used as a loading control.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry

    C14orf166 upregulation in human bladder cancer cell lines. a Quantitative RT-PCR determination of C14orf166 expression in immortalized human bladder epithelial SV-HUC-1 cells and bladder cancer J82, UM3, RT4, 5637, and T24 cells. Transcription levels were normalized to GAPDH expression. b Western blot determination of C14orf166 expression in SV-HUC-1, J82, UM3, RT4, 5637, and T24 cells. α-Tubulin was used as the loading control. * P

    Journal: Journal of Translational Medicine

    Article Title: C14orf166 is a high-risk biomarker for bladder cancer and promotes bladder cancer cell proliferation

    doi: 10.1186/s12967-016-0801-4

    Figure Lengend Snippet: C14orf166 upregulation in human bladder cancer cell lines. a Quantitative RT-PCR determination of C14orf166 expression in immortalized human bladder epithelial SV-HUC-1 cells and bladder cancer J82, UM3, RT4, 5637, and T24 cells. Transcription levels were normalized to GAPDH expression. b Western blot determination of C14orf166 expression in SV-HUC-1, J82, UM3, RT4, 5637, and T24 cells. α-Tubulin was used as the loading control. * P

    Article Snippet: An antibody to human α-Tubulin (1:5000, Santa Cruz) was used as a loading control.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot

    C14orf166 regulated the key proteins that regulate G1/S transition. a Cell cycle assay of the effect of C14orf166 knockdown on cell cycle progression. b Quantitative RT-PCR determination of Cyclin D1, P21, and P27 expression after C14orf166 knockdown in RT4 cells. Transcription levels were normalized to GAPDH expression. c Western blot determination of Cyclin D1, p21, p27, Rb, and p-Rb expression in RT4 cells. α-Tubulin was used as the loading control. d Quantitative RT-PCR determination of Cyclin D1, P21 and P27 expression following C14orf166 knockdown in T24 cells. Transcription levels were normalized to GAPDH expression. e Western blot determination of Cyclin D1, p21, p27, Rb, and p-Rb expression in T24 cells. α-Tubulin was used as the loading control. * P

    Journal: Journal of Translational Medicine

    Article Title: C14orf166 is a high-risk biomarker for bladder cancer and promotes bladder cancer cell proliferation

    doi: 10.1186/s12967-016-0801-4

    Figure Lengend Snippet: C14orf166 regulated the key proteins that regulate G1/S transition. a Cell cycle assay of the effect of C14orf166 knockdown on cell cycle progression. b Quantitative RT-PCR determination of Cyclin D1, P21, and P27 expression after C14orf166 knockdown in RT4 cells. Transcription levels were normalized to GAPDH expression. c Western blot determination of Cyclin D1, p21, p27, Rb, and p-Rb expression in RT4 cells. α-Tubulin was used as the loading control. d Quantitative RT-PCR determination of Cyclin D1, P21 and P27 expression following C14orf166 knockdown in T24 cells. Transcription levels were normalized to GAPDH expression. e Western blot determination of Cyclin D1, p21, p27, Rb, and p-Rb expression in T24 cells. α-Tubulin was used as the loading control. * P

    Article Snippet: An antibody to human α-Tubulin (1:5000, Santa Cruz) was used as a loading control.

    Techniques: Cell Cycle Assay, Quantitative RT-PCR, Expressing, Western Blot

    ZLN005 upregulates PGC-1 α and its downstream mitochondrial gene targets in ARPE-19 cells. (a) Relative gene expression (RE) of PGC-1α in ARPE-19 treated for 48 hours with 10 μ M ZLN005 (ZLN, n = 5) compared to vehicle (veh, n = 4). (b) Representative protein blot of PGC-1 α and α -tubulin bands and (c) quantification showed upregulation of PGC-1 α protein expression with ZLN005 treatment (all conditions, n = 4). (d) RPE-specific genes, BEST1 and RLBP1 , are upregulated with ZLN005 treatment (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (e) Treatment with 10 μ M ZLN005 increases the mitochondrial dynamic gene, MFN1 , and replication genes, TFAM and POLG (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (f, g) These changes do not impact mitochondrial morphology, as observed through (f) MitoTracker imaging (red) and (g) quantification in median fluorescence intensity (MFI) per mm 2 (Unt, n = 3; veh, n = 2; ZLN, n = 3). All gene expression data were analyzed using Student's t -test. Statistical significance is represented as follows: ns P > 0.05, ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Pharmaceutical Induction of PGC-1α Promotes Retinal Pigment Epithelial Cell Metabolism and Protects against Oxidative Damage

    doi: 10.1155/2018/9248640

    Figure Lengend Snippet: ZLN005 upregulates PGC-1 α and its downstream mitochondrial gene targets in ARPE-19 cells. (a) Relative gene expression (RE) of PGC-1α in ARPE-19 treated for 48 hours with 10 μ M ZLN005 (ZLN, n = 5) compared to vehicle (veh, n = 4). (b) Representative protein blot of PGC-1 α and α -tubulin bands and (c) quantification showed upregulation of PGC-1 α protein expression with ZLN005 treatment (all conditions, n = 4). (d) RPE-specific genes, BEST1 and RLBP1 , are upregulated with ZLN005 treatment (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (e) Treatment with 10 μ M ZLN005 increases the mitochondrial dynamic gene, MFN1 , and replication genes, TFAM and POLG (veh, n = 4 for all genes; ZLN, n = 5 for all genes). (f, g) These changes do not impact mitochondrial morphology, as observed through (f) MitoTracker imaging (red) and (g) quantification in median fluorescence intensity (MFI) per mm 2 (Unt, n = 3; veh, n = 2; ZLN, n = 3). All gene expression data were analyzed using Student's t -test. Statistical significance is represented as follows: ns P > 0.05, ∗ P

    Article Snippet: The membranes were incubated overnight at 4°C with mouse anti-human PGC-1 α (1 : 250, EMD Millipore) and mouse anti-human α -tubulin (1 : 1000, Cell Signaling, Danvers, MA, USA).

    Techniques: Pyrolysis Gas Chromatography, Expressing, Imaging, Fluorescence

    Rearrangement of the cytoskeleton and evolution of mechanical properties. a At the beginning of NETosis F-actin (red) is laterally enriched and localizes in the lamellipodia. α-Tubulin filaments (green) are arranged originating from the microtubule organizing center (MTOC) in unstimulated cells. Within the next hours (during P1) cytoskeletal components disintegrate. Remaining F-actin accumulates at the cell margin and α-tubulin is first rearranged in centrosome-like structures which disappear at the beginning of P2. CLSM images of fixed cells. Activation = PMA (100 nM). Blue = chromatin. Scale = 10 µm. b , c Inhibition of NET formation with the F-actin polymerization inhibitors Cytochalasin D (100 nM) and Latrunculin A (1 µM), F-actin-stabilizing drug Jasplakinolide (10 µM) and the ROCK-inhibitor Y-27632 (19.2 µM) significantly reduces the formation of NETs (measured as %-relative number of decondensed nuclei after 180 min compared to activation with PMA only) in P1, while P2 depends less or not on F-actin stabilization and ROCK-inhibition. Statistics: two-way ANOVA (Bonferroni’s multiple comparisons test; * p

    Journal: Nature Communications

    Article Title: Chromatin swelling drives neutrophil extracellular trap release

    doi: 10.1038/s41467-018-06263-5

    Figure Lengend Snippet: Rearrangement of the cytoskeleton and evolution of mechanical properties. a At the beginning of NETosis F-actin (red) is laterally enriched and localizes in the lamellipodia. α-Tubulin filaments (green) are arranged originating from the microtubule organizing center (MTOC) in unstimulated cells. Within the next hours (during P1) cytoskeletal components disintegrate. Remaining F-actin accumulates at the cell margin and α-tubulin is first rearranged in centrosome-like structures which disappear at the beginning of P2. CLSM images of fixed cells. Activation = PMA (100 nM). Blue = chromatin. Scale = 10 µm. b , c Inhibition of NET formation with the F-actin polymerization inhibitors Cytochalasin D (100 nM) and Latrunculin A (1 µM), F-actin-stabilizing drug Jasplakinolide (10 µM) and the ROCK-inhibitor Y-27632 (19.2 µM) significantly reduces the formation of NETs (measured as %-relative number of decondensed nuclei after 180 min compared to activation with PMA only) in P1, while P2 depends less or not on F-actin stabilization and ROCK-inhibition. Statistics: two-way ANOVA (Bonferroni’s multiple comparisons test; * p

    Article Snippet: Subsequently cells were stained with monoclonal anti-human MPO (IgG, mouse, 1:500) (ab25989, Abcam), monoclonal anti-human α-Tubulin (IgG, rabbit, 1:50) (#2125, Cell Signaling Technology) or polyclonal anti-human lamin B1 (IgG, rabbit, 1:1000) (ab16048, Abcam) as primary antibodies over night (4 °C) and visualized with polyclonal anti-mouse Alexa488 (IgG, goat, 1:1000) (#4408, Cell Signaling Technology) or polyclonal anti-rabbit Alexa488 (IgG, goat, 1:500) (A-11034, ThermoFisher Scientific) as secondary antibodies.

    Techniques: Confocal Laser Scanning Microscopy, Activation Assay, Inhibition

    SmCBP1 and SmGCN5 play a key role in Smp14 expression and female sexual reproductive development. A dsRNAi experiment was carried out on sixteen adult worm pairs cultivated for seven days. Worms were electroporated and soaked with dsRNAi from SmCBP1 or SmGCN5 or with the non-specific dsRNAi LUC. On the seventh day of culture, the mRNA levels of SmCBP1, SmGCN5 or Smp14 (graphs A and B) were determined by qRT-PCR, normalized by the α-tubulin transcript levels. The results are depicted in relation to the non-specific dsRNAi (dsLUC) mRNA levels. The effects of the dsRNAi on targeted-protein synthesis were assessed by Western blot (panels C and D). The total number of eggs laid by the same parasites that received the dsRNAi was counted (graphs E and F). Adult worms from the dsRNAi experiments were analyzed by confocal laser scanning microscopy (G). Worms received either the control dsRNAi LUC (panels a and d) or the dsRNAi for SmCBP1 (panels b and e) or SmGCN5 (panels c and f). Panels a–c, and d–f, reveal the effect of the dsRNAi in the ovary, and vitellaria, respectively. OV: ovary; mo: mature oocytes; io: immature oocytes; v: vitellaria; rs: receptaculum seminis. Results are pooled from three independent experiments. Western blots were repeated three times. The confocal microscopy images are representative of several parasites analyzed. Scale bars 10 µm. Student's t-test was applied, with *p

    Journal: PLoS Pathogens

    Article Title: Epigenetic Changes Modulate Schistosome Egg Formation and Are a Novel Target for Reducing Transmission of Schistosomiasis

    doi: 10.1371/journal.ppat.1004116

    Figure Lengend Snippet: SmCBP1 and SmGCN5 play a key role in Smp14 expression and female sexual reproductive development. A dsRNAi experiment was carried out on sixteen adult worm pairs cultivated for seven days. Worms were electroporated and soaked with dsRNAi from SmCBP1 or SmGCN5 or with the non-specific dsRNAi LUC. On the seventh day of culture, the mRNA levels of SmCBP1, SmGCN5 or Smp14 (graphs A and B) were determined by qRT-PCR, normalized by the α-tubulin transcript levels. The results are depicted in relation to the non-specific dsRNAi (dsLUC) mRNA levels. The effects of the dsRNAi on targeted-protein synthesis were assessed by Western blot (panels C and D). The total number of eggs laid by the same parasites that received the dsRNAi was counted (graphs E and F). Adult worms from the dsRNAi experiments were analyzed by confocal laser scanning microscopy (G). Worms received either the control dsRNAi LUC (panels a and d) or the dsRNAi for SmCBP1 (panels b and e) or SmGCN5 (panels c and f). Panels a–c, and d–f, reveal the effect of the dsRNAi in the ovary, and vitellaria, respectively. OV: ovary; mo: mature oocytes; io: immature oocytes; v: vitellaria; rs: receptaculum seminis. Results are pooled from three independent experiments. Western blots were repeated three times. The confocal microscopy images are representative of several parasites analyzed. Scale bars 10 µm. Student's t-test was applied, with *p

    Article Snippet: Philip T. LoVerde) and human α-tubulin (Abcam).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Confocal Laser Scanning Microscopy, Confocal Microscopy

    HAT inhibition impairs Smp14 transcription and translation in S. mansoni adult worms. Ten adult worm pairs were cultivated with increasing concentrations of the HAT inhibitor PU139 or DMSO as vehicle. PU139 concentrations between 0.01 and 20 µM were solubilized in 0.1% DMSO, and 50 µM PU139 was only soluble in 0.5% DMSO. (A) qRT-PCR analysis of Smp14 mRNA levels after a two-day incubation time. (B) Western blot analysis of total protein extract from a two-day worm pair culture using polyclonal antibodies against Smp14 (upper panels) and monoclonal antibodies against α-tubulin (lower panels) as loading control. (C) qRT-PCR of Smp14 after a longer period (four days) of PU139 (20 µM) treatment. (D) Smp14 protein levels at the fourth day of treatment with 20 µM PU139. Graphs are pooled from three independent experiments. Student's t-test was applied, with *p

    Journal: PLoS Pathogens

    Article Title: Epigenetic Changes Modulate Schistosome Egg Formation and Are a Novel Target for Reducing Transmission of Schistosomiasis

    doi: 10.1371/journal.ppat.1004116

    Figure Lengend Snippet: HAT inhibition impairs Smp14 transcription and translation in S. mansoni adult worms. Ten adult worm pairs were cultivated with increasing concentrations of the HAT inhibitor PU139 or DMSO as vehicle. PU139 concentrations between 0.01 and 20 µM were solubilized in 0.1% DMSO, and 50 µM PU139 was only soluble in 0.5% DMSO. (A) qRT-PCR analysis of Smp14 mRNA levels after a two-day incubation time. (B) Western blot analysis of total protein extract from a two-day worm pair culture using polyclonal antibodies against Smp14 (upper panels) and monoclonal antibodies against α-tubulin (lower panels) as loading control. (C) qRT-PCR of Smp14 after a longer period (four days) of PU139 (20 µM) treatment. (D) Smp14 protein levels at the fourth day of treatment with 20 µM PU139. Graphs are pooled from three independent experiments. Student's t-test was applied, with *p

    Article Snippet: Philip T. LoVerde) and human α-tubulin (Abcam).

    Techniques: HAT Assay, Inhibition, Quantitative RT-PCR, Incubation, Western Blot

    MTs are twisting around IFs in TnT as observed with confocal microscopy. ( a ) Positions 1–5 correspond to ( b ) x-z and y-z projections and show that MTs (α-tubulin, green) rotate around IF core (CK7, red). Note the rotation of MTs (arrowheads) in x-z and in y-z. ( c ) Gray values of MTs (α-tubulin) and IFs (CK7) fluorescence intensity along TnT are plotted. Gray values of α-tubulin reach point 0 between two peaks, which correspond to one twist of MTs around IFs. Presented normal urothelial cells were methanol fixed.

    Journal: Scientific Reports

    Article Title: Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

    doi: 10.1038/s41598-018-35370-y

    Figure Lengend Snippet: MTs are twisting around IFs in TnT as observed with confocal microscopy. ( a ) Positions 1–5 correspond to ( b ) x-z and y-z projections and show that MTs (α-tubulin, green) rotate around IF core (CK7, red). Note the rotation of MTs (arrowheads) in x-z and in y-z. ( c ) Gray values of MTs (α-tubulin) and IFs (CK7) fluorescence intensity along TnT are plotted. Gray values of α-tubulin reach point 0 between two peaks, which correspond to one twist of MTs around IFs. Presented normal urothelial cells were methanol fixed.

    Article Snippet: For double immunolabelling of α-tubulin – the subunit of MTs and cytokeratin 7 (CK7) – from the keratin subfamily of IFs, urothelial cells were fixed in 100% ice-cold methanol for 5 min washed in PBS and left in blocking buffer (1% bovine serum albumin and 10% goat serum in PBS) for 1 h. Afterwards the cells were incubated with rabbit polyclonal anti-human α-tubulin (Abcam 15246, 1:20) and mouse monoclonal anti-human CK7 (M7018 Dako, 1:20) primary antibodies for 1 h at 37 °C.

    Techniques: Confocal Microscopy, Fluorescence

    Helical organization of MTs in TnT as observed with 3D-SIM microscopy. ( a , b ) Maximal intensity profile of x-y projections of MT signal (α-tubulin, green) and IF signal (CK7, red) at the initiation segment and the attaching segment of TnT. Dashed white region is × 1.5 enlarged in ( a′ ) and represents x-y projection with helical MT organization ( a″ ). Gray values on graph correspond to the fluorescence intensity signal of MTs (α-tubulin) along yellow line. Eight white arrowheads correspond to the eight peaks on graph and indicate four MT twists. Presented normal urothelial cells were fixed with methanol.

    Journal: Scientific Reports

    Article Title: Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

    doi: 10.1038/s41598-018-35370-y

    Figure Lengend Snippet: Helical organization of MTs in TnT as observed with 3D-SIM microscopy. ( a , b ) Maximal intensity profile of x-y projections of MT signal (α-tubulin, green) and IF signal (CK7, red) at the initiation segment and the attaching segment of TnT. Dashed white region is × 1.5 enlarged in ( a′ ) and represents x-y projection with helical MT organization ( a″ ). Gray values on graph correspond to the fluorescence intensity signal of MTs (α-tubulin) along yellow line. Eight white arrowheads correspond to the eight peaks on graph and indicate four MT twists. Presented normal urothelial cells were fixed with methanol.

    Article Snippet: For double immunolabelling of α-tubulin – the subunit of MTs and cytokeratin 7 (CK7) – from the keratin subfamily of IFs, urothelial cells were fixed in 100% ice-cold methanol for 5 min washed in PBS and left in blocking buffer (1% bovine serum albumin and 10% goat serum in PBS) for 1 h. Afterwards the cells were incubated with rabbit polyclonal anti-human α-tubulin (Abcam 15246, 1:20) and mouse monoclonal anti-human CK7 (M7018 Dako, 1:20) primary antibodies for 1 h at 37 °C.

    Techniques: Microscopy, Fluorescence

    Characterization of TnTs. ( a - a′ ) Arrowheads denote TnTs of living/unfixed normal and cancer urothelial cells. ( b ) Actin filaments (F-actin, green) in normal urothelial cells. Panel b shows four sequential optical sections with relative z-positions (in µm) and indicates that TnT (arrowheads) does not adhere to the substratum. ( c - c″ ) Arrows denote TnTs of normal urothelial cells with MTs (α-tubulin, green) and IFs (CK7, red) co-labelling, on merged ( c ) and separate images ( c′ - c″ ). Images were obtained with phase-contrast and wide-field fluorescence microscope with ApoTome device. Panel (b) was performed after paraformaldehyde fixation and ( c ) after methanol fixation, both of normal urothelial cells.

    Journal: Scientific Reports

    Article Title: Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

    doi: 10.1038/s41598-018-35370-y

    Figure Lengend Snippet: Characterization of TnTs. ( a - a′ ) Arrowheads denote TnTs of living/unfixed normal and cancer urothelial cells. ( b ) Actin filaments (F-actin, green) in normal urothelial cells. Panel b shows four sequential optical sections with relative z-positions (in µm) and indicates that TnT (arrowheads) does not adhere to the substratum. ( c - c″ ) Arrows denote TnTs of normal urothelial cells with MTs (α-tubulin, green) and IFs (CK7, red) co-labelling, on merged ( c ) and separate images ( c′ - c″ ). Images were obtained with phase-contrast and wide-field fluorescence microscope with ApoTome device. Panel (b) was performed after paraformaldehyde fixation and ( c ) after methanol fixation, both of normal urothelial cells.

    Article Snippet: For double immunolabelling of α-tubulin – the subunit of MTs and cytokeratin 7 (CK7) – from the keratin subfamily of IFs, urothelial cells were fixed in 100% ice-cold methanol for 5 min washed in PBS and left in blocking buffer (1% bovine serum albumin and 10% goat serum in PBS) for 1 h. Afterwards the cells were incubated with rabbit polyclonal anti-human α-tubulin (Abcam 15246, 1:20) and mouse monoclonal anti-human CK7 (M7018 Dako, 1:20) primary antibodies for 1 h at 37 °C.

    Techniques: Fluorescence, Microscopy

    Ars2 is commonly expressed in glioblastoma cells. (A) Real-time qPCR analysis of Ars2 mRNA expression levels in four glioblastoma cell lines (U87, A172, LN-229 and U251) and one normal astrocyte cell line (HEB). The data represent the average obtained from three independent experiments and are presented as the mean ± SD. (B) Western blot analysis of Ars2 expression in four glioblastoma cell lines and one normal astrocyte cell line. α-tubulin is shown as the loading control. (C) Immunohistochemical analysis of Ars2 expression in human glioblastoma tumor and paired normal tissues. Scale bar, 10 µm. (D) Statistical analyses of immunohistochemical results of Ars2 expression levels in 12 paired samples of normal and glioblastoma tissues; P=0.0001. Ars2, arsenic resistance protein 2.

    Journal: Oncology Reports

    Article Title: Knockdown of arsenic resistance protein 2 inhibits human glioblastoma cell proliferation through the MAPK/ERK pathway

    doi: 10.3892/or.2018.6777

    Figure Lengend Snippet: Ars2 is commonly expressed in glioblastoma cells. (A) Real-time qPCR analysis of Ars2 mRNA expression levels in four glioblastoma cell lines (U87, A172, LN-229 and U251) and one normal astrocyte cell line (HEB). The data represent the average obtained from three independent experiments and are presented as the mean ± SD. (B) Western blot analysis of Ars2 expression in four glioblastoma cell lines and one normal astrocyte cell line. α-tubulin is shown as the loading control. (C) Immunohistochemical analysis of Ars2 expression in human glioblastoma tumor and paired normal tissues. Scale bar, 10 µm. (D) Statistical analyses of immunohistochemical results of Ars2 expression levels in 12 paired samples of normal and glioblastoma tissues; P=0.0001. Ars2, arsenic resistance protein 2.

    Article Snippet: The primary antibodies were as follows: Rabbit anti-human Ars2 (dilution 1:2,000; cat. no. ab192999; Abcam, Cambridge, UK), mouse anti-human α-tubulin (dilution 1:2,000; cat. no. B-5-1-2; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), rabbit anti-human CDK2 (dilution 1:1,000; cat. no. 2546S; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-human CDK4 (dilution 1:1,000; cat. no. 12790S; Cell Signaling Technology), rabbit anti-human cyclin D1 (dilution 1:1,000; cat. no. 2922S; Cell Signaling Technology), rabbit anti-human cyclin E2 (dilution 1:1,000; cat. no. ab40890; Abcam), rabbit anti-human p21 (dilution 1:1,000; cat. no. ab109199; Abcam), rabbit anti-human PAK2 (dilution 1:1,000; cat. no. 2608S; Cell Signaling Technology), rabbit anti-human ERK1/2 (dilution 1:500; cat. no. 4695S; Cell Signaling Technology), rabbit anti-human phospho-ERK1/2 (Thr202/Tyr204) (dilution 1:2,000; cat. no. 4376S; Cell Signaling Technology), rabbit anti-human MEK1/2 (dilution 1:1,000; cat. no. 9122S; Cell Signaling Technology), and rabbit anti-human phospho-MEK1/2 (Ser217/221) (1:500; cat. no. 9121S; Cell Signaling Technology).

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

    Downregulation of Ars2 inhibits glioblastoma cell proliferation. (A) Top panels: Western blot analysis of Ars2 expression in glioblastoma cells with GFP knockdown or Ars2 knockdown; α-tubulin is shown as the loading control. Lower panels: Real-time qPCR analysis of Ars2 mRNA expression levels in glioblastoma cells with GFP knockdown or Ars2 knockdown. (B) Left panels: Morphologic examination of glioblastoma cells with GFP knockdown or Ars2 knockdown. Right panels: Cell counting analysis with trypan blue dye staining. (C) Cell growth curves of glioblastoma cells with GFP knockdown or Ars2 knockdown were analyzed by MTT assay. For all data in A-C, each value represents the average obtained from three independent experiments, and the data are presented as the mean ± SD (error bars). Statistical analyses were performed using two-tailed Student's t-tests, ***P

    Journal: Oncology Reports

    Article Title: Knockdown of arsenic resistance protein 2 inhibits human glioblastoma cell proliferation through the MAPK/ERK pathway

    doi: 10.3892/or.2018.6777

    Figure Lengend Snippet: Downregulation of Ars2 inhibits glioblastoma cell proliferation. (A) Top panels: Western blot analysis of Ars2 expression in glioblastoma cells with GFP knockdown or Ars2 knockdown; α-tubulin is shown as the loading control. Lower panels: Real-time qPCR analysis of Ars2 mRNA expression levels in glioblastoma cells with GFP knockdown or Ars2 knockdown. (B) Left panels: Morphologic examination of glioblastoma cells with GFP knockdown or Ars2 knockdown. Right panels: Cell counting analysis with trypan blue dye staining. (C) Cell growth curves of glioblastoma cells with GFP knockdown or Ars2 knockdown were analyzed by MTT assay. For all data in A-C, each value represents the average obtained from three independent experiments, and the data are presented as the mean ± SD (error bars). Statistical analyses were performed using two-tailed Student's t-tests, ***P

    Article Snippet: The primary antibodies were as follows: Rabbit anti-human Ars2 (dilution 1:2,000; cat. no. ab192999; Abcam, Cambridge, UK), mouse anti-human α-tubulin (dilution 1:2,000; cat. no. B-5-1-2; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), rabbit anti-human CDK2 (dilution 1:1,000; cat. no. 2546S; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-human CDK4 (dilution 1:1,000; cat. no. 12790S; Cell Signaling Technology), rabbit anti-human cyclin D1 (dilution 1:1,000; cat. no. 2922S; Cell Signaling Technology), rabbit anti-human cyclin E2 (dilution 1:1,000; cat. no. ab40890; Abcam), rabbit anti-human p21 (dilution 1:1,000; cat. no. ab109199; Abcam), rabbit anti-human PAK2 (dilution 1:1,000; cat. no. 2608S; Cell Signaling Technology), rabbit anti-human ERK1/2 (dilution 1:500; cat. no. 4695S; Cell Signaling Technology), rabbit anti-human phospho-ERK1/2 (Thr202/Tyr204) (dilution 1:2,000; cat. no. 4376S; Cell Signaling Technology), rabbit anti-human MEK1/2 (dilution 1:1,000; cat. no. 9122S; Cell Signaling Technology), and rabbit anti-human phospho-MEK1/2 (Ser217/221) (1:500; cat. no. 9121S; Cell Signaling Technology).

    Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Cell Counting, Staining, MTT Assay, Two Tailed Test

    PDAC microtumours are triggered by a cell-in-cell structure anchored to a microisland and are covered by membranous expression of α-tubulin. ( a ). ( b ). ( c ) When a mix of CFSE-labelled PCI-55 cells and unlabelled PCI-55 cells were sparsely cultured, they showed a cell-in-cell structure on the rough circular microisland. ( d ) Papillary PCI-55 microtumour formed by cell-in-cell structures. ( e , f ) Immunofluorescent staining for α-tubulin in the microtumours self-organised by CFSE-labelled PCI-55 cells. CFSE High -labelled PCI-55 cells penetrating CFSE Low -labelled cells that formed a microtumour ( e ). Multinucleated giant PCI-55 cells containing at least six high CFSE-labelled PCI-55 cells ( f , g ). The surface of a papillary microtumour self-organised by PCI-55 cells. Bars: 30 µm. ( h ) Many PCI-55 cells in a mitotic phase were outside the surface of the microtumours. Z-stack full-focus images of immunofluorescent staining for α-tubulin in PCI-55 microtumours anchored to the micro/nanoplate. ( i ) Counts of mitotic cells in PCI-55 microtumours. ( j , k ) ( j ). 3D images ( k ). ( l ). m, Immunofluorescent staining for α-tubulin in PCI-55 cells colonised on the peritoneum. CFSE-labelled PCI-55 cells were grafted intraperitoneally into SCID mice. After 3 days, the peritonea were extracted.

    Journal: Scientific Reports

    Article Title: Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion

    doi: 10.1038/s41598-018-32122-w

    Figure Lengend Snippet: PDAC microtumours are triggered by a cell-in-cell structure anchored to a microisland and are covered by membranous expression of α-tubulin. ( a ). ( b ). ( c ) When a mix of CFSE-labelled PCI-55 cells and unlabelled PCI-55 cells were sparsely cultured, they showed a cell-in-cell structure on the rough circular microisland. ( d ) Papillary PCI-55 microtumour formed by cell-in-cell structures. ( e , f ) Immunofluorescent staining for α-tubulin in the microtumours self-organised by CFSE-labelled PCI-55 cells. CFSE High -labelled PCI-55 cells penetrating CFSE Low -labelled cells that formed a microtumour ( e ). Multinucleated giant PCI-55 cells containing at least six high CFSE-labelled PCI-55 cells ( f , g ). The surface of a papillary microtumour self-organised by PCI-55 cells. Bars: 30 µm. ( h ) Many PCI-55 cells in a mitotic phase were outside the surface of the microtumours. Z-stack full-focus images of immunofluorescent staining for α-tubulin in PCI-55 microtumours anchored to the micro/nanoplate. ( i ) Counts of mitotic cells in PCI-55 microtumours. ( j , k ) ( j ). 3D images ( k ). ( l ). m, Immunofluorescent staining for α-tubulin in PCI-55 cells colonised on the peritoneum. CFSE-labelled PCI-55 cells were grafted intraperitoneally into SCID mice. After 3 days, the peritonea were extracted.

    Article Snippet: Mouse anti-human α-tubulin (clone DM1A, eBioscience), mouse anti-human dynein intermediate chain 1 (Abcam, Cambridge, UK) monoclonal antibodies (mAbs) were used for immunofluorescence staining.

    Techniques: Expressing, Cell Culture, Staining, Mouse Assay

    Membranous expression of α-tubulin in human PDAC specimens. ( a,b ) Histological analysis of human PDAC specimens. ( a , b ) Cell-in-cell structures and membranous α-tubulin expression in human PDAC specimens. HE staining and immunostaining for α-tubulin in a PanIN lesion from PDAC patient 1 ( a ). Normal pancreatic ducts and PanIN (upper panels), and invasive PDAC (lower panels) in PDAC Patient 2 ( b ). N: normal pancreatic ducts, M: malignant lesion in pancreatic ducts. HE staining and immunostaining for α-tubulin in invasive PDAC from PDAC patient 2 ( c ). Normal pancreatic ducts and PanIN (left panels), and invasive PDAC (right panels) in PDAC patient 3 ( d ). Invasive PDAC from PDAC patients 4, 5 and 6 ( e ). Arrow heads indicate cell-in-cell structures formed by live cells and arrows indicate cell-in-cell structures formed by dead cells.

    Journal: Scientific Reports

    Article Title: Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion

    doi: 10.1038/s41598-018-32122-w

    Figure Lengend Snippet: Membranous expression of α-tubulin in human PDAC specimens. ( a,b ) Histological analysis of human PDAC specimens. ( a , b ) Cell-in-cell structures and membranous α-tubulin expression in human PDAC specimens. HE staining and immunostaining for α-tubulin in a PanIN lesion from PDAC patient 1 ( a ). Normal pancreatic ducts and PanIN (upper panels), and invasive PDAC (lower panels) in PDAC Patient 2 ( b ). N: normal pancreatic ducts, M: malignant lesion in pancreatic ducts. HE staining and immunostaining for α-tubulin in invasive PDAC from PDAC patient 2 ( c ). Normal pancreatic ducts and PanIN (left panels), and invasive PDAC (right panels) in PDAC patient 3 ( d ). Invasive PDAC from PDAC patients 4, 5 and 6 ( e ). Arrow heads indicate cell-in-cell structures formed by live cells and arrows indicate cell-in-cell structures formed by dead cells.

    Article Snippet: Mouse anti-human α-tubulin (clone DM1A, eBioscience), mouse anti-human dynein intermediate chain 1 (Abcam, Cambridge, UK) monoclonal antibodies (mAbs) were used for immunofluorescence staining.

    Techniques: Expressing, Staining, Immunostaining