progressive deletions Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher pmir report vector
    HLA-G 3’UTR polymorphisms. (A) Variations in HLA-G mRNA (red) along the 3’UTR. The less frequent variants are positioned below the most frequent one in the nucleotide sequence. Polymorphic sites are framed in bold when the frequency of the minor allele worldwide is higher than 1%. Purple single arrows point to the three predicted polyadenylation signals. Vertical lines with horizontal arrows indicate the 5’ end of the HLA-G nucleotide sequences for 1Fter, 2R and 4R primers used in <t>pMIR-REPORT</t> ™ constructions. (B) Sequence comparison of the most frequent 3’UTR haplotypes (21 worldwide populations; 2n = 3870) [ 25 ] analyzed in the present study. Frequencies are indicated on the right part. UTR-1 and UTR-2 differ at 5 positions (pink rectangles). UTR-6 and UTR-18 (analyzed in the present work) only differ at one position and depending on the UTR typing strategy that was used (i.e., targeting or not +3227 SNP), UTR-18 could be confused with UTR-6 in several studies.
    Pmir Report Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmir report vector/product/Thermo Fisher
    Average 99 stars, based on 4287 article reviews
    Price from $9.99 to $1999.99
    pmir report vector - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore 6 ohda
    Nitrite administration ameliorates pathology in rotenone and <t>6-OHDA</t> rat models of PD . (A) Schematic of the experimental paradigm for nitrite and rotenone administration. (B) Representative image of striatal TH immunohistochemistry reveals severe depletion in DA fibers after rotenone exposure, which is strongly mitigated on treatment with inorganic nitrite (scale bar: 1 mm). (C) Representative image of rotenone-induced DA loss in the SNpc, which is prevented by nitrite co-treatment (scale bar: 100 μm). (D) Quantification of striatal innervation in (B) by densitometry. (E) Quantification of DA loss in the SNpc (C) by unbiased stereological counts. (F) Experimental 6-OHDA setup: In the acute paradigm ( top ), nitrite was administered 24 and 1 h before stereotactic infusion in the rat striatum; in the chronic paradigm ( bottom ), nitrite was administered orally, dissolved in drinking water, starting 7 days after induction of the lesion. (G, H) Nitrite administration ameliorates the 6-OHDA-induced lesions in striata and in SNpc. (I) Representative images of striata cross-sections injected with 6-OHDA ( ipsilateral ) and the respective untreated side ( contralateral ) of nitrite-treated rats (Scale bar: 1 mm) (* p
    6 Ohda, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/6 ohda/product/Millipore
    Average 99 stars, based on 1860 article reviews
    Price from $9.99 to $1999.99
    6 ohda - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    97
    Cell Signaling Technology Inc mycn
    <t>MYC,</t> MYCL and <t>MYCN</t> inhibition by Omomyc induces cell cycle arrest through the activation of p21, in some cases through the TP73 pathway
    Mycn, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mycn/product/Cell Signaling Technology Inc
    Average 97 stars, based on 92 article reviews
    Price from $9.99 to $1999.99
    mycn - by Bioz Stars, 2020-09
    97/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc ndrg1
    Increase of expression of neuroendocrine phenotypic marker by epidrug (5-Aza) in PCa cells PCa cells (PC3 or DU145) (2 × 10 5 ) were seeded in 10cm culture dishes. The cells were treated by vehicle or 5-Aza (5µM) for 10 days. ( A–C ) mRNA levels of N-Myc downstream regulated 1 <t>(NDRG1),</t> Enolase-2 (ENO2), and Synaptophysin expression in vehicle or 5-Aza (5µM) treated PCa cells (PC3 or DU145) as quantified by real-time PCR. The data in Fig. 2A–C are representative of mean ± s.d (n=6). P values were calculated by Student’s t -test. ( D ) Protein levels of NDRG1, ENO2, and Synaptophysin following vehicle or 5-Aza (5µM) treatments in PCa cells (PC3 or DU145) as quantified by Western blots.
    Ndrg1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ndrg1/product/Cell Signaling Technology Inc
    Average 93 stars, based on 117 article reviews
    Price from $9.99 to $1999.99
    ndrg1 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    TaKaRa deletion kit
    Increase of expression of neuroendocrine phenotypic marker by epidrug (5-Aza) in PCa cells PCa cells (PC3 or DU145) (2 × 10 5 ) were seeded in 10cm culture dishes. The cells were treated by vehicle or 5-Aza (5µM) for 10 days. ( A–C ) mRNA levels of N-Myc downstream regulated 1 <t>(NDRG1),</t> Enolase-2 (ENO2), and Synaptophysin expression in vehicle or 5-Aza (5µM) treated PCa cells (PC3 or DU145) as quantified by real-time PCR. The data in Fig. 2A–C are representative of mean ± s.d (n=6). P values were calculated by Student’s t -test. ( D ) Protein levels of NDRG1, ENO2, and Synaptophysin following vehicle or 5-Aza (5µM) treatments in PCa cells (PC3 or DU145) as quantified by Western blots.
    Deletion Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deletion kit/product/TaKaRa
    Average 93 stars, based on 142 article reviews
    Price from $9.99 to $1999.99
    deletion kit - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc mfn2
    MitoQ ameliorates IDD development in an ex vivo model. The rat IVDs from compression group were cultured under compression treatment for 2 wk. The rat IVDs from MitoQ group were cultured under compression and MitoQ cotreatment for 2 wk. (A) HE and SO staining of the rat IVD tissues. (B) The histological scores of the rat IVD tissues according to histological grading scale. (C‐D) TUNEL staining and fluorescence microscope analysis were used to evaluate the apoptosis in the rat IVD tissues. Scale bar: 100 μm. (E) The content of H 2 O 2 in the disc samples. (F‐H) The levels of caspase‐3, c‐caspase 3, Drp1, <t>Mfn2,</t> PINK1, Parkin, P62, Nrf2 and LC3 proteins in the rat IVD tissues were measured by Western blotting. Data are represented as the mean ± SD. *** P
    Mfn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mfn2/product/Cell Signaling Technology Inc
    Average 99 stars, based on 357 article reviews
    Price from $9.99 to $1999.99
    mfn2 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher pmir report luciferase vector
    MitoQ ameliorates IDD development in an ex vivo model. The rat IVDs from compression group were cultured under compression treatment for 2 wk. The rat IVDs from MitoQ group were cultured under compression and MitoQ cotreatment for 2 wk. (A) HE and SO staining of the rat IVD tissues. (B) The histological scores of the rat IVD tissues according to histological grading scale. (C‐D) TUNEL staining and fluorescence microscope analysis were used to evaluate the apoptosis in the rat IVD tissues. Scale bar: 100 μm. (E) The content of H 2 O 2 in the disc samples. (F‐H) The levels of caspase‐3, c‐caspase 3, Drp1, <t>Mfn2,</t> PINK1, Parkin, P62, Nrf2 and LC3 proteins in the rat IVD tissues were measured by Western blotting. Data are represented as the mean ± SD. *** P
    Pmir Report Luciferase Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmir report luciferase vector/product/Thermo Fisher
    Average 94 stars, based on 3112 article reviews
    Price from $9.99 to $1999.99
    pmir report luciferase vector - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    96
    Cell Signaling Technology Inc anti ndrg1
    BRD9 is part of the SWI-SNF complex and binds MYC enhancer sequences. ( A ) Immunoblots showing Co-IP of BRD9 with the SWI-SNF subunit BRG1 from MCF-10A nuclear extracts or ( B ) nuclear extracts isolated from DKI cells transduced with lenti-BRD9. ( C ) ChIP data (Mean ± SD) showing BRD9 occupancy at MYC super-enhancer and VIM promoter DNA regions. BRD4 and SUMO2 represent positive control sequences; NEG represents a non-specific negative control sequence. ( D ) RT-qPCR (Mean ± SD) of MYC transcript in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. ( E ) Immunoblot data showing MYC (with densitometric quantification, lower panel), target <t>NDRG1,</t> and VIM expression in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. **, p
    Anti Ndrg1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ndrg1/product/Cell Signaling Technology Inc
    Average 96 stars, based on 52 article reviews
    Price from $9.99 to $1999.99
    anti ndrg1 - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    90
    Funakoshi lymperopoulos a
    BRD9 is part of the SWI-SNF complex and binds MYC enhancer sequences. ( A ) Immunoblots showing Co-IP of BRD9 with the SWI-SNF subunit BRG1 from MCF-10A nuclear extracts or ( B ) nuclear extracts isolated from DKI cells transduced with lenti-BRD9. ( C ) ChIP data (Mean ± SD) showing BRD9 occupancy at MYC super-enhancer and VIM promoter DNA regions. BRD4 and SUMO2 represent positive control sequences; NEG represents a non-specific negative control sequence. ( D ) RT-qPCR (Mean ± SD) of MYC transcript in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. ( E ) Immunoblot data showing MYC (with densitometric quantification, lower panel), target <t>NDRG1,</t> and VIM expression in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. **, p
    Lymperopoulos A, supplied by Funakoshi, used in various techniques. Bioz Stars score: 90/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lymperopoulos a/product/Funakoshi
    Average 90 stars, based on 129 article reviews
    Price from $9.99 to $1999.99
    lymperopoulos a - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc anti phospho ndrg1 thr346
    BRD9 is part of the SWI-SNF complex and binds MYC enhancer sequences. ( A ) Immunoblots showing Co-IP of BRD9 with the SWI-SNF subunit BRG1 from MCF-10A nuclear extracts or ( B ) nuclear extracts isolated from DKI cells transduced with lenti-BRD9. ( C ) ChIP data (Mean ± SD) showing BRD9 occupancy at MYC super-enhancer and VIM promoter DNA regions. BRD4 and SUMO2 represent positive control sequences; NEG represents a non-specific negative control sequence. ( D ) RT-qPCR (Mean ± SD) of MYC transcript in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. ( E ) Immunoblot data showing MYC (with densitometric quantification, lower panel), target <t>NDRG1,</t> and VIM expression in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. **, p
    Anti Phospho Ndrg1 Thr346, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho ndrg1 thr346/product/Cell Signaling Technology Inc
    Average 95 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    anti phospho ndrg1 thr346 - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology sox4
    Loss of <t>SOX4</t> significantly inhibits PTEN deletion-induced prostate tumorigenesis. ( A ) Representative images of prostate glands from wild-type, Pten −/− and Pten −/− /Sox4 −/− mice are shown. Homozygous deletion
    Sox4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sox4/product/Santa Cruz Biotechnology
    Average 93 stars, based on 506 article reviews
    Price from $9.99 to $1999.99
    sox4 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    95
    Promega pgl3 basic vector
    CYP6D1v1 promoter sequence from −365 to −267 (from LPR) is able to mediate PB induction. Promoter constructs are numbered relative to the transcription start site (TSS) at +1. Relative luciferase activity was estimated by normalizing each signal of each promoter construct to the mean of signals of <t>pGL3-Basic</t> vector in the same replicate. Bars represent the mean relative luciferase activity ± S.D. of three independent transfections. Gray bars represent the signal in the presence of PB and white bars represent the control. Double asterisks indicate a greater PB induction relative to the next shorter promoter construct (p
    Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 27770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/Promega
    Average 95 stars, based on 27770 article reviews
    Price from $9.99 to $1999.99
    pgl3 basic vector - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    93
    TaKaRa exonuclease iii
    Detection of the sites activated by E2F. ( A ) <t>Three</t> regions of the p68 gene promoter (+9/+16, –11/–3 and –19/–12) were mutated (the mutations were transversions of each base in the three base stretch and are indicated by X). These sites are represented by hatched boxes. Each mutant plasmid was co-transfected into NIH 3T3 cells with a plasmid carrying CMV-E2F-1 or CMV. The promoter activities are indicated as relative activity with respect to the activity of <t>pKL12</t> without E2F-1. The promoter activity with and without E2F-1 is represented by open and closed bars, respectively. The increase in the relative activity of luciferase with CMV-E2F-1 is presented on the right. ( B ) The wild-type sequence and mutation sequence. Open boxes indicate three homologous E2F-binding sites and arrows represent transversion mutations.
    Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease iii/product/TaKaRa
    Average 93 stars, based on 174 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology smad4
    Activation of mTOR and increased TGFβ1 ligand in murine SGTs. (A) Immunohistochemistry staining of p-mTOR (brown) in SPAs developed from Pten SG-KO mice ( n = 7) or <t>Smad4</t> SG-KO mice ( n = 5), and SACCs developed from Pten.Smad4 SG-KO mice ( n = 6). WT: wild-type. Scale bar: 50 μm. (B) ELISA measurement of TGFβ1 levels in SPAs developed from Pten SG-KO mice or Smad4 SG-KO mice, and SACCs developed from Pten.Smad4 SG-KO mice. n = 3 of each group. * P
    Smad4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/smad4/product/Santa Cruz Biotechnology
    Average 93 stars, based on 2340 article reviews
    Price from $9.99 to $1999.99
    smad4 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher steponeplus real time pcr system
    Activation of mTOR and increased TGFβ1 ligand in murine SGTs. (A) Immunohistochemistry staining of p-mTOR (brown) in SPAs developed from Pten SG-KO mice ( n = 7) or <t>Smad4</t> SG-KO mice ( n = 5), and SACCs developed from Pten.Smad4 SG-KO mice ( n = 6). WT: wild-type. Scale bar: 50 μm. (B) ELISA measurement of TGFβ1 levels in SPAs developed from Pten SG-KO mice or Smad4 SG-KO mice, and SACCs developed from Pten.Smad4 SG-KO mice. n = 3 of each group. * P
    Steponeplus Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/steponeplus real time pcr system/product/Thermo Fisher
    Average 99 stars, based on 52235 article reviews
    Price from $9.99 to $1999.99
    steponeplus real time pcr system - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Addgene inc nfatc1
    Activation of mTOR and increased TGFβ1 ligand in murine SGTs. (A) Immunohistochemistry staining of p-mTOR (brown) in SPAs developed from Pten SG-KO mice ( n = 7) or <t>Smad4</t> SG-KO mice ( n = 5), and SACCs developed from Pten.Smad4 SG-KO mice ( n = 6). WT: wild-type. Scale bar: 50 μm. (B) ELISA measurement of TGFβ1 levels in SPAs developed from Pten SG-KO mice or Smad4 SG-KO mice, and SACCs developed from Pten.Smad4 SG-KO mice. n = 3 of each group. * P
    Nfatc1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nfatc1/product/Addgene inc
    Average 92 stars, based on 69 article reviews
    Price from $9.99 to $1999.99
    nfatc1 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    93
    Aviva Systems twist1
    <t>Twist1</t> deletion in basal keratinocytes suppresses TPA-induced epidermal proliferation
    Twist1, supplied by Aviva Systems, used in various techniques. Bioz Stars score: 93/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/twist1/product/Aviva Systems
    Average 93 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    twist1 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc p15
    Functional characterization of CDKI variants. a Protein expression in whole cell lysates of HEK293 cells transfected with wild type or mutagenized cDNA expression constructs. Top panel was probed with the indicated specific anti-CDKI antibody, mid dle panel with anti-NPTII antibody (to assess transfection efficiency), and lower panel with anti-tubulin antibody (to assess protein loading). Relative level of wild-type (wt) and mutant protein expression is shown ( n =3), with wt protein level=1. b Protein level in nuclear and cytoplasmic extracts of HEK293 cells transfected with wild-type or mutagenized cDNA expression constructs. Top panels of cytoplasmic extract (CE), and bottom panels of nuclear extract (NE) were probed with the indicated specific anti-CDKI antibody, anti-tubulin (to assess protein loading in CE, and to assess the efficiency of sub-cellular fractionation in NE), or with anti-p84 (to assess protein loading in the NE, and to assess the efficiency of sub-cellular fractionation in the CE). Relative level of wt and mutant protein expression in CE and NE is shown ( n =3), with wt protein level=1, * p =0.04. c In vitro translated (IVT) proteins used for GST pull-down assay in ‘ D ’. 35 S-Methionine labeled wild-type or mutagenized proteins were generated by in vitro translation from cDNA expression constructs. IVT proteins were detected by SDS-PAGE and autoradiography. d Protein-binding to CDK2 or CDK6. GST pull-down assay for binding to GST, GST-CDK2, or GST-CDK6 of 35 S-methionine labeled IVT wt or mutagenized proteins. The bound IVT protein was detected by SDS-PAGE and autoradiography. Input lane contained 10 % of the IVT product incubated with GST, GST-CDK2, or GST-CDK6. Relative binding of wt and mutant protein to GST-fusion proteins is shown with wt protein binding= 1 ( n =3 for <t>p15</t> and p18; n =2 for p21), ** p
    P15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p15/product/Cell Signaling Technology Inc
    Average 94 stars, based on 232 article reviews
    Price from $9.99 to $1999.99
    p15 - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc phospho ndrg1
    <t>p-NDRG1</t> is able to predict the outcome of EGFR -mutant NSCLC patients. (A and B) GSEA revealed an enrichment of glycolytic genes in patients with EGFR -mutant NSCLC tumors with high p-NDRG1 expression (A) and an enrichment of oxidative phosphorylation genes in EGFR -mutant NSCLC patients with low p-NDRG1 expression (B). NES, normalized enrichment score. FDR, false discovery rate. (C) Correlation between p-NDRG1 expression and overall survival (OS) in NSCLC patients with EGFR driver mutations. The reverse phase protein array (RPPA) data for the relevant patients were downloaded from The Cancer Proteome Atlas (TCGA) database. The X-axis shows batch-normalized protein expression levels of p-NDRG1. The survival time of each patient is shown in the Y-axis in months. Black line denotes the linear fit.
    Phospho Ndrg1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho ndrg1/product/Cell Signaling Technology Inc
    Average 93 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    phospho ndrg1 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    HLA-G 3’UTR polymorphisms. (A) Variations in HLA-G mRNA (red) along the 3’UTR. The less frequent variants are positioned below the most frequent one in the nucleotide sequence. Polymorphic sites are framed in bold when the frequency of the minor allele worldwide is higher than 1%. Purple single arrows point to the three predicted polyadenylation signals. Vertical lines with horizontal arrows indicate the 5’ end of the HLA-G nucleotide sequences for 1Fter, 2R and 4R primers used in pMIR-REPORT ™ constructions. (B) Sequence comparison of the most frequent 3’UTR haplotypes (21 worldwide populations; 2n = 3870) [ 25 ] analyzed in the present study. Frequencies are indicated on the right part. UTR-1 and UTR-2 differ at 5 positions (pink rectangles). UTR-6 and UTR-18 (analyzed in the present work) only differ at one position and depending on the UTR typing strategy that was used (i.e., targeting or not +3227 SNP), UTR-18 could be confused with UTR-6 in several studies.

    Journal: PLoS ONE

    Article Title: Haplotypes of the HLA-G 3’ Untranslated Region Respond to Endogenous Factors of HLA-G+ and HLA-G- Cell Lines Differentially

    doi: 10.1371/journal.pone.0169032

    Figure Lengend Snippet: HLA-G 3’UTR polymorphisms. (A) Variations in HLA-G mRNA (red) along the 3’UTR. The less frequent variants are positioned below the most frequent one in the nucleotide sequence. Polymorphic sites are framed in bold when the frequency of the minor allele worldwide is higher than 1%. Purple single arrows point to the three predicted polyadenylation signals. Vertical lines with horizontal arrows indicate the 5’ end of the HLA-G nucleotide sequences for 1Fter, 2R and 4R primers used in pMIR-REPORT ™ constructions. (B) Sequence comparison of the most frequent 3’UTR haplotypes (21 worldwide populations; 2n = 3870) [ 25 ] analyzed in the present study. Frequencies are indicated on the right part. UTR-1 and UTR-2 differ at 5 positions (pink rectangles). UTR-6 and UTR-18 (analyzed in the present work) only differ at one position and depending on the UTR typing strategy that was used (i.e., targeting or not +3227 SNP), UTR-18 could be confused with UTR-6 in several studies.

    Article Snippet: Reporter constructs For each 3’UTR haplotype (UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-18, and UTR-7) two constructions were performed with the pMIR-REPORT™ vector (Ambion® , ThermoFisher Scientific): the putative HLA-G polyadenylation signals were kept (1Fter-2R primer set used) or not (1Fter-4R primer set used), thereby maintaining or deleting the polymorphic sites at positions +3187, +3196, and +3227 ( ).

    Techniques: Sequencing

    Nitrite administration ameliorates pathology in rotenone and 6-OHDA rat models of PD . (A) Schematic of the experimental paradigm for nitrite and rotenone administration. (B) Representative image of striatal TH immunohistochemistry reveals severe depletion in DA fibers after rotenone exposure, which is strongly mitigated on treatment with inorganic nitrite (scale bar: 1 mm). (C) Representative image of rotenone-induced DA loss in the SNpc, which is prevented by nitrite co-treatment (scale bar: 100 μm). (D) Quantification of striatal innervation in (B) by densitometry. (E) Quantification of DA loss in the SNpc (C) by unbiased stereological counts. (F) Experimental 6-OHDA setup: In the acute paradigm ( top ), nitrite was administered 24 and 1 h before stereotactic infusion in the rat striatum; in the chronic paradigm ( bottom ), nitrite was administered orally, dissolved in drinking water, starting 7 days after induction of the lesion. (G, H) Nitrite administration ameliorates the 6-OHDA-induced lesions in striata and in SNpc. (I) Representative images of striata cross-sections injected with 6-OHDA ( ipsilateral ) and the respective untreated side ( contralateral ) of nitrite-treated rats (Scale bar: 1 mm) (* p

    Journal: Antioxidants & Redox Signaling

    Article Title: Mitochondrial Complex I Reversible S-Nitrosation Improves Bioenergetics and Is Protective in Parkinson's Disease

    doi: 10.1089/ars.2017.6992

    Figure Lengend Snippet: Nitrite administration ameliorates pathology in rotenone and 6-OHDA rat models of PD . (A) Schematic of the experimental paradigm for nitrite and rotenone administration. (B) Representative image of striatal TH immunohistochemistry reveals severe depletion in DA fibers after rotenone exposure, which is strongly mitigated on treatment with inorganic nitrite (scale bar: 1 mm). (C) Representative image of rotenone-induced DA loss in the SNpc, which is prevented by nitrite co-treatment (scale bar: 100 μm). (D) Quantification of striatal innervation in (B) by densitometry. (E) Quantification of DA loss in the SNpc (C) by unbiased stereological counts. (F) Experimental 6-OHDA setup: In the acute paradigm ( top ), nitrite was administered 24 and 1 h before stereotactic infusion in the rat striatum; in the chronic paradigm ( bottom ), nitrite was administered orally, dissolved in drinking water, starting 7 days after induction of the lesion. (G, H) Nitrite administration ameliorates the 6-OHDA-induced lesions in striata and in SNpc. (I) Representative images of striata cross-sections injected with 6-OHDA ( ipsilateral ) and the respective untreated side ( contralateral ) of nitrite-treated rats (Scale bar: 1 mm) (* p

    Article Snippet: Sodium nitrite (563218), MPP+ (D048), MPTP (M0896), Carboxy-PTIO potassium salt (NO-scavenger, C221), NOC-18 (NO-donor, A5581), 6-OHDA (H4381), oligomycin (75351), FCCP (C2920), rotenone (557368), antimycin (A8674), N-Ethylmaleimide (NEM, E3876), digitonin (D141), mannitol (M4125), glutamate (1446600), malate (4694 U), trigonelline (T5509), and 5,5-Dimethyl-1,3-cyclohexanedione (dimedone, D153303) were obtained from Sigma-Aldrich (St. Louis, MO).

    Techniques: Immunohistochemistry, Injection

    Rotational Behavior and Histological Evaluation of Hemi-Parkinsonian Rat Models that Are Grafted iPSC-Derived DA Progenitors and Received E2B Treatment (A and B) Methamphetamine-induced rotational behavior (A) and improvement ratio of rotational behavior (B) in 6-OHDA-lesioned rats after cell grafting. Quantitative data are represented as the mean ± SD (n = 9–12 independent animals, pre to 12 weeks; n = 5–7 independent animals, 14 and 16 weeks). Significance (two-way ANOVA with Tukey's multiple comparisons test): ∗ p

    Journal: Stem Cell Reports

    Article Title: Estradiol Facilitates Functional Integration of iPSC-Derived Dopaminergic Neurons into Striatal Neuronal Circuits via Activation of Integrin α5β1

    doi: 10.1016/j.stemcr.2016.02.008

    Figure Lengend Snippet: Rotational Behavior and Histological Evaluation of Hemi-Parkinsonian Rat Models that Are Grafted iPSC-Derived DA Progenitors and Received E2B Treatment (A and B) Methamphetamine-induced rotational behavior (A) and improvement ratio of rotational behavior (B) in 6-OHDA-lesioned rats after cell grafting. Quantitative data are represented as the mean ± SD (n = 9–12 independent animals, pre to 12 weeks; n = 5–7 independent animals, 14 and 16 weeks). Significance (two-way ANOVA with Tukey's multiple comparisons test): ∗ p

    Article Snippet: Hemi-Parkinsonian Rat Model and Rotational Behavior Female 10-week-old X-SCID rats were used for the 6-OHDA-lesioned hemi-parkinsonian model. A total of 15 μg of 6-OHDA hydrochloride (Sigma) in 3 μl of saline with 0.02% AA was stereotactically injected into the medial forebrain bundle in the left side of the rat brain (from the bregma: A −5.3, L +1.2, V −7.0, and TB +1.5).

    Techniques: Derivative Assay

    MYC, MYCL and MYCN inhibition by Omomyc induces cell cycle arrest through the activation of p21, in some cases through the TP73 pathway

    Journal: Oncotarget

    Article Title: Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation

    doi: 10.18632/oncotarget.8826

    Figure Lengend Snippet: MYC, MYCL and MYCN inhibition by Omomyc induces cell cycle arrest through the activation of p21, in some cases through the TP73 pathway

    Article Snippet: Lysates (15-30μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the following antibodies: MYC (sc-40, Santa Cruz), MYCL (#AF4050, R & D), MYCN (#9405, Cell Signaling), Omomyc, p21 (#2947, Cell Signaling), p27 (sc-1641, Santa Cruz), p16 (51-1325GR, BD), PARP1 (#9542, Cell Signaling), TP73 (sc-7957, Santa Cruz), α-Tubulin (CP06, CalBiochemicals).

    Techniques: Inhibition, Activation Assay

    Omomyc induces growth suppression in SCLC cells A. Status of the MYC family genes, TP53 , and RB1 in SCLC cell lines used in this study. mut: mutated. Predominant type of the cell cycle arrest, occurrence of apoptosis and levels of p21, p27 and p16 after MYC inhibition by Omomyc are shown. B. Immunoblot analysis for the expression of MYC, MYCL or MYCN in SCLC cells. Media were changed 24 hr before collection of the cells. C. Growth curve of SCLC cells in the presence or absence of doxycycline (DX). Cumulative population doubling level (PDL) was calculated by adding the PDLs of the previous passages. Data are shown as the mean ± SD of four counts from a single representative experiment. P-values were calculated by unpaired two-tailed t-test. *p

    Journal: Oncotarget

    Article Title: Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation

    doi: 10.18632/oncotarget.8826

    Figure Lengend Snippet: Omomyc induces growth suppression in SCLC cells A. Status of the MYC family genes, TP53 , and RB1 in SCLC cell lines used in this study. mut: mutated. Predominant type of the cell cycle arrest, occurrence of apoptosis and levels of p21, p27 and p16 after MYC inhibition by Omomyc are shown. B. Immunoblot analysis for the expression of MYC, MYCL or MYCN in SCLC cells. Media were changed 24 hr before collection of the cells. C. Growth curve of SCLC cells in the presence or absence of doxycycline (DX). Cumulative population doubling level (PDL) was calculated by adding the PDLs of the previous passages. Data are shown as the mean ± SD of four counts from a single representative experiment. P-values were calculated by unpaired two-tailed t-test. *p

    Article Snippet: Lysates (15-30μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the following antibodies: MYC (sc-40, Santa Cruz), MYCL (#AF4050, R & D), MYCN (#9405, Cell Signaling), Omomyc, p21 (#2947, Cell Signaling), p27 (sc-1641, Santa Cruz), p16 (51-1325GR, BD), PARP1 (#9542, Cell Signaling), TP73 (sc-7957, Santa Cruz), α-Tubulin (CP06, CalBiochemicals).

    Techniques: Inhibition, Expressing, Two Tailed Test

    CIP2A, I2PP2A, and PP2A in neuroblastoma cell lines. (A) Immunoblotting revealed CIP2A, I2PP2A, and PP2A expression in all four neuroblastoma cell lines studied. There were no differences in expression between the MYCN nonamplified SH-EP and the isogenic MYCN amplified WAC2 cells. (B) SK-N-AS and SH-EP neuroblastoma cells ( MYCN nonamplified) were transfected with MYCN overexpression vector and cell lysates examined for I2PP2A and PP2A. MYCN was successfully expressed in both cell lines. Expression of I2PP2A and PP2A was not affected by MYCN overexpression. (C). Neuroblastoma cell lines were treated with siRNA knockdown of I2PP2A. Whole cell lysates revealed knockdown of I2PP2A with no change in PP2A expression. (D) Neuroblastoma cell lines were treated with siRNA knockdown of CIP2A. Whole cell lysates revealed knockdown of CIP2A with no change in PP2A expression. (E) PP2A activity was measured in SK-N-AS cells following siRNA inhibition. Inhibition of I2PP2A, CIP2A, or dual inhibition led to significant increases in PP2A activity.

    Journal: Translational Oncology

    Article Title: Investigation of PP2A and Its Endogenous Inhibitors in Neuroblastoma Cell Survival and Tumor Growth

    doi: 10.1016/j.tranon.2018.09.011

    Figure Lengend Snippet: CIP2A, I2PP2A, and PP2A in neuroblastoma cell lines. (A) Immunoblotting revealed CIP2A, I2PP2A, and PP2A expression in all four neuroblastoma cell lines studied. There were no differences in expression between the MYCN nonamplified SH-EP and the isogenic MYCN amplified WAC2 cells. (B) SK-N-AS and SH-EP neuroblastoma cells ( MYCN nonamplified) were transfected with MYCN overexpression vector and cell lysates examined for I2PP2A and PP2A. MYCN was successfully expressed in both cell lines. Expression of I2PP2A and PP2A was not affected by MYCN overexpression. (C). Neuroblastoma cell lines were treated with siRNA knockdown of I2PP2A. Whole cell lysates revealed knockdown of I2PP2A with no change in PP2A expression. (D) Neuroblastoma cell lines were treated with siRNA knockdown of CIP2A. Whole cell lysates revealed knockdown of CIP2A with no change in PP2A expression. (E) PP2A activity was measured in SK-N-AS cells following siRNA inhibition. Inhibition of I2PP2A, CIP2A, or dual inhibition led to significant increases in PP2A activity.

    Article Snippet: Primary antibodies used for Western blotting included the following: anti-I2PP2A (H-120) (sc-25564) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-PP2A (ab32104) and anti-CIP2A (ab99518) from Abcam (Cambridge, MA), anti–total AKT (9272), anti–phospho-AKT (S473; 9271), anti-MYCN (9405), p44/42 MAP Kinase [ERK1/2 (9102)], anti–phospho-p44/42 MAPK [phospho-ERK, T202/T204, (4377)] from Cell Signaling Technology (Danvers, MA), and anti–β-actin from Sigma (A1978, Sigma Aldrich, St. Louis, MO).

    Techniques: Expressing, Amplification, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Inhibition

    ) and MYCN-expressing SH-SY5Y neuroblastoma cell lines. (A) Western blot analysis of N-myc protein expression in neuroblastoma cells after 72 h of transfection. β-actin was used for the equal protein loading of the cell lysates. (B) Semi-quantitative RT-PCR analysis of MYCN mRNA expression in neuroblastoma cells after 72 h of transfection. GAPDH was used as a loading control. Data are expressed as a ratio of arbitary pixel density units.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Antitumor effects of imatinib mesylate and synergistic cytotoxicity with an arsenic compound in neuroblastoma cell lines

    doi: 10.3892/etm.2011.220

    Figure Lengend Snippet: ) and MYCN-expressing SH-SY5Y neuroblastoma cell lines. (A) Western blot analysis of N-myc protein expression in neuroblastoma cells after 72 h of transfection. β-actin was used for the equal protein loading of the cell lysates. (B) Semi-quantitative RT-PCR analysis of MYCN mRNA expression in neuroblastoma cells after 72 h of transfection. GAPDH was used as a loading control. Data are expressed as a ratio of arbitary pixel density units.

    Article Snippet: The antibodies used were anti-MYCN Ab (#9405; Cell Signaling Technology) and β-actin Ab (Sigma).

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

    Increase of expression of neuroendocrine phenotypic marker by epidrug (5-Aza) in PCa cells PCa cells (PC3 or DU145) (2 × 10 5 ) were seeded in 10cm culture dishes. The cells were treated by vehicle or 5-Aza (5µM) for 10 days. ( A–C ) mRNA levels of N-Myc downstream regulated 1 (NDRG1), Enolase-2 (ENO2), and Synaptophysin expression in vehicle or 5-Aza (5µM) treated PCa cells (PC3 or DU145) as quantified by real-time PCR. The data in Fig. 2A–C are representative of mean ± s.d (n=6). P values were calculated by Student’s t -test. ( D ) Protein levels of NDRG1, ENO2, and Synaptophysin following vehicle or 5-Aza (5µM) treatments in PCa cells (PC3 or DU145) as quantified by Western blots.

    Journal: Journal of cellular biochemistry

    Article Title: Reduction of Histone Marks, H3K9me3 and H3K27me3 by Epidrug Induces Neuroendocrine Differentiation in Prostate Cancer

    doi: 10.1002/jcb.26586

    Figure Lengend Snippet: Increase of expression of neuroendocrine phenotypic marker by epidrug (5-Aza) in PCa cells PCa cells (PC3 or DU145) (2 × 10 5 ) were seeded in 10cm culture dishes. The cells were treated by vehicle or 5-Aza (5µM) for 10 days. ( A–C ) mRNA levels of N-Myc downstream regulated 1 (NDRG1), Enolase-2 (ENO2), and Synaptophysin expression in vehicle or 5-Aza (5µM) treated PCa cells (PC3 or DU145) as quantified by real-time PCR. The data in Fig. 2A–C are representative of mean ± s.d (n=6). P values were calculated by Student’s t -test. ( D ) Protein levels of NDRG1, ENO2, and Synaptophysin following vehicle or 5-Aza (5µM) treatments in PCa cells (PC3 or DU145) as quantified by Western blots.

    Article Snippet: Primary antibodies used were as follows: N-Myc downstream regulated 1 (NDRG1, 1:1,000 dilution, cat. 9485, Cell Signaling,), Synaptophysin (1:1,000 dilution, cat. 4329, Cell Signaling,), Enolase-2 (ENO2, 1:1,000 dilution, cat. 9536, Cell Signaling,), H3K9me3 (1:1,000 dilution, cat. 13969, Cell Signaling), and H3K27me3 (1:1,000 dilution, cat. 9733, Cell Signaling).

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

    Reduction of histone marks on the NDRG1 promoter by epidrug (5-Aza) in PCa cells ( A ) Examination of global changes of histone silent marks, H3K9me3 and H3K27me3 in PCa cells (PC3, DU145) following in vehicle or 5-Aza treated PCa cells by Western blots. ( B ) Experimental design of CHIP qPCR assays in vehicle or 5-Aza treated PCa cells. ( C ) A scheme shows the primer site at the promoter region from transcription start site of the human NDRG1 genomic locus. ( D ) % respective input of H3K9me3 and H3K27me3 levels at the NDRG1 genomic locus as determined by CHIP qPCR assays in vehicle or 5-Aza treated PCa cells (PC3, DU145). The data in Fig. 4D are representative of mean ± s.d. (n=3). P values are calculated by Student’s t -test. ( E ) A summary diagram shows the activation of chromatin by histone demethylation of H3K9me3 and H3K27me3 following 5-Aza treatment, resulting in the increase of NDRG1 transcription.

    Journal: Journal of cellular biochemistry

    Article Title: Reduction of Histone Marks, H3K9me3 and H3K27me3 by Epidrug Induces Neuroendocrine Differentiation in Prostate Cancer

    doi: 10.1002/jcb.26586

    Figure Lengend Snippet: Reduction of histone marks on the NDRG1 promoter by epidrug (5-Aza) in PCa cells ( A ) Examination of global changes of histone silent marks, H3K9me3 and H3K27me3 in PCa cells (PC3, DU145) following in vehicle or 5-Aza treated PCa cells by Western blots. ( B ) Experimental design of CHIP qPCR assays in vehicle or 5-Aza treated PCa cells. ( C ) A scheme shows the primer site at the promoter region from transcription start site of the human NDRG1 genomic locus. ( D ) % respective input of H3K9me3 and H3K27me3 levels at the NDRG1 genomic locus as determined by CHIP qPCR assays in vehicle or 5-Aza treated PCa cells (PC3, DU145). The data in Fig. 4D are representative of mean ± s.d. (n=3). P values are calculated by Student’s t -test. ( E ) A summary diagram shows the activation of chromatin by histone demethylation of H3K9me3 and H3K27me3 following 5-Aza treatment, resulting in the increase of NDRG1 transcription.

    Article Snippet: Primary antibodies used were as follows: N-Myc downstream regulated 1 (NDRG1, 1:1,000 dilution, cat. 9485, Cell Signaling,), Synaptophysin (1:1,000 dilution, cat. 4329, Cell Signaling,), Enolase-2 (ENO2, 1:1,000 dilution, cat. 9536, Cell Signaling,), H3K9me3 (1:1,000 dilution, cat. 13969, Cell Signaling), and H3K27me3 (1:1,000 dilution, cat. 9733, Cell Signaling).

    Techniques: Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activation Assay

    Association of H3K9me3 and H3K27me3 reduction with NDRG1 in s.c. PCa tumors and human primary PCa tumors ( A ) NDRG1 (green) expression (white arrow) in H3K9me3 (red) or H3K27me3 (red) reduced cells in vehicle or 5-Aza pretreated s.c. PCa tumors as detected by immunofluorescence co-staining. Blue, DAPI nuclear stain. Bar=20µm. ( B ) Quantification of Fig. 5A. Data represent as mean ± s.d. (n=8/group) (Student’s t -test). ( C ) NDRG1 (green) expression (white arrow) in H3K9me3 (red) or H3K27me3 (red) expressing cells in human primary PCa tumors from the tissue microarray samples (TMA) from PCa patients as detected by immunofluorescence co-staining. Blue, DAPI nuclear stain. Bar=20µm. TMAs are normal prostate tissue (n=8), Gleason 6 prostate cancer tissue (n=9), and Gleason 9 prostate cancer tissue (n=5). ( D ) Quantification of Fig. 5C. Data represent as mean ± s.d. (Student’s t -test).

    Journal: Journal of cellular biochemistry

    Article Title: Reduction of Histone Marks, H3K9me3 and H3K27me3 by Epidrug Induces Neuroendocrine Differentiation in Prostate Cancer

    doi: 10.1002/jcb.26586

    Figure Lengend Snippet: Association of H3K9me3 and H3K27me3 reduction with NDRG1 in s.c. PCa tumors and human primary PCa tumors ( A ) NDRG1 (green) expression (white arrow) in H3K9me3 (red) or H3K27me3 (red) reduced cells in vehicle or 5-Aza pretreated s.c. PCa tumors as detected by immunofluorescence co-staining. Blue, DAPI nuclear stain. Bar=20µm. ( B ) Quantification of Fig. 5A. Data represent as mean ± s.d. (n=8/group) (Student’s t -test). ( C ) NDRG1 (green) expression (white arrow) in H3K9me3 (red) or H3K27me3 (red) expressing cells in human primary PCa tumors from the tissue microarray samples (TMA) from PCa patients as detected by immunofluorescence co-staining. Blue, DAPI nuclear stain. Bar=20µm. TMAs are normal prostate tissue (n=8), Gleason 6 prostate cancer tissue (n=9), and Gleason 9 prostate cancer tissue (n=5). ( D ) Quantification of Fig. 5C. Data represent as mean ± s.d. (Student’s t -test).

    Article Snippet: Primary antibodies used were as follows: N-Myc downstream regulated 1 (NDRG1, 1:1,000 dilution, cat. 9485, Cell Signaling,), Synaptophysin (1:1,000 dilution, cat. 4329, Cell Signaling,), Enolase-2 (ENO2, 1:1,000 dilution, cat. 9536, Cell Signaling,), H3K9me3 (1:1,000 dilution, cat. 13969, Cell Signaling), and H3K27me3 (1:1,000 dilution, cat. 9733, Cell Signaling).

    Techniques: Expressing, Immunofluorescence, Staining, Microarray

    MitoQ ameliorates IDD development in an ex vivo model. The rat IVDs from compression group were cultured under compression treatment for 2 wk. The rat IVDs from MitoQ group were cultured under compression and MitoQ cotreatment for 2 wk. (A) HE and SO staining of the rat IVD tissues. (B) The histological scores of the rat IVD tissues according to histological grading scale. (C‐D) TUNEL staining and fluorescence microscope analysis were used to evaluate the apoptosis in the rat IVD tissues. Scale bar: 100 μm. (E) The content of H 2 O 2 in the disc samples. (F‐H) The levels of caspase‐3, c‐caspase 3, Drp1, Mfn2, PINK1, Parkin, P62, Nrf2 and LC3 proteins in the rat IVD tissues were measured by Western blotting. Data are represented as the mean ± SD. *** P

    Journal: Cell Proliferation

    Article Title: The mitochondria‐targeted anti‐oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance. The mitochondria‐targeted anti‐oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance

    doi: 10.1111/cpr.12779

    Figure Lengend Snippet: MitoQ ameliorates IDD development in an ex vivo model. The rat IVDs from compression group were cultured under compression treatment for 2 wk. The rat IVDs from MitoQ group were cultured under compression and MitoQ cotreatment for 2 wk. (A) HE and SO staining of the rat IVD tissues. (B) The histological scores of the rat IVD tissues according to histological grading scale. (C‐D) TUNEL staining and fluorescence microscope analysis were used to evaluate the apoptosis in the rat IVD tissues. Scale bar: 100 μm. (E) The content of H 2 O 2 in the disc samples. (F‐H) The levels of caspase‐3, c‐caspase 3, Drp1, Mfn2, PINK1, Parkin, P62, Nrf2 and LC3 proteins in the rat IVD tissues were measured by Western blotting. Data are represented as the mean ± SD. *** P

    Article Snippet: For immunohistochemical analysis, sections were incubated with primary antibodies against cleaved caspase‐3 (#9664, Cell Signaling Technology), Drp1 (ab56788, Abcam), Mfn2 (#9482, Cell Signaling Technology), Parkin (ab77924, Abcam), LC3 (#3868, Cell Signaling Technology), P62 (ab56416, Abcam) and Nrf2 (ab137550, Abcam) at 4°C overnight and then with the appropriate secondary antibodies.

    Techniques: Ex Vivo, Cell Culture, Staining, TUNEL Assay, Fluorescence, Microscopy, Western Blot

    Effects of MitoQ on compression‐induced alterations in mitochondrial dynamics in human NP cells. (A‐B) Human NP cells were pre‐treated with MitoQ (500 nmol/L) for 2 h and then exposed to compression for 36 h. The colocalization of Drp1 and Tom20 was examined by confocal microscopy. Scale bar: 10 μm. (C‐D) The protein levels of mitochondrial Drp1 (mito‐Drp1) and cytoplasmic Drp1 (cyto‐Drp1) in the human NP cells were measured by Western blotting. (D‐F) The protein levels of total Drp1, Mff, Fis1, Mid49, Mid51, Mfn1, Mfn2 and Opa1 in the human NP cells were measured by Western blotting. Data are represented as the mean ± SD. *** P

    Journal: Cell Proliferation

    Article Title: The mitochondria‐targeted anti‐oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance. The mitochondria‐targeted anti‐oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance

    doi: 10.1111/cpr.12779

    Figure Lengend Snippet: Effects of MitoQ on compression‐induced alterations in mitochondrial dynamics in human NP cells. (A‐B) Human NP cells were pre‐treated with MitoQ (500 nmol/L) for 2 h and then exposed to compression for 36 h. The colocalization of Drp1 and Tom20 was examined by confocal microscopy. Scale bar: 10 μm. (C‐D) The protein levels of mitochondrial Drp1 (mito‐Drp1) and cytoplasmic Drp1 (cyto‐Drp1) in the human NP cells were measured by Western blotting. (D‐F) The protein levels of total Drp1, Mff, Fis1, Mid49, Mid51, Mfn1, Mfn2 and Opa1 in the human NP cells were measured by Western blotting. Data are represented as the mean ± SD. *** P

    Article Snippet: For immunohistochemical analysis, sections were incubated with primary antibodies against cleaved caspase‐3 (#9664, Cell Signaling Technology), Drp1 (ab56788, Abcam), Mfn2 (#9482, Cell Signaling Technology), Parkin (ab77924, Abcam), LC3 (#3868, Cell Signaling Technology), P62 (ab56416, Abcam) and Nrf2 (ab137550, Abcam) at 4°C overnight and then with the appropriate secondary antibodies.

    Techniques: Confocal Microscopy, Western Blot

    Analysis of mitophagy in Mfn2 KO U2OS cells. ( A ) Genomic sequence of human Mfn2 (exon 3) that was mutated in U2OS cells using CRISPR/Cas9. The arrow indicates the codon corresponding to methionine-1; leucine-29 (‘L29’), lysine-30 (‘K30’) and the introduced stop codon (‘*’) are also indicated. ( B ) Immunoblot analysis of whole-cell lysates from Mfn2 KO clones (A4 and A5). ( C ) Mitochondrial morphology in Mfn2 KO cells, as revealed by confocal imaging of TOM20 (green) staining. The asterisks indicate nuclei. Scale bar, 20 microns. ( D ) Representative wide-field images of mitochondrial polarization in live WT and Mfn2 KO (clone A4) cells as indicated by TMRM staining. ( E ) Representative confocal images of GFP-parkin recruitment to mitochondria as a function of time in WT or Mfn2 KO (clone A4) U2OS cells, transfected with GFP-parkin and treated with 20 μM CCCP. Red asterisks indicate cells in which GFP-parkin has fully translocated to mitochondria. Scale bar, 20 microns. ( F ) Quantification of parkin recruitment in cells from ( E ). Data points represent mean ± SEM, n = 3 replicates per condition, with > 100 cells counted per condition for each replicate. n.s., not significant; *, p

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: Analysis of mitophagy in Mfn2 KO U2OS cells. ( A ) Genomic sequence of human Mfn2 (exon 3) that was mutated in U2OS cells using CRISPR/Cas9. The arrow indicates the codon corresponding to methionine-1; leucine-29 (‘L29’), lysine-30 (‘K30’) and the introduced stop codon (‘*’) are also indicated. ( B ) Immunoblot analysis of whole-cell lysates from Mfn2 KO clones (A4 and A5). ( C ) Mitochondrial morphology in Mfn2 KO cells, as revealed by confocal imaging of TOM20 (green) staining. The asterisks indicate nuclei. Scale bar, 20 microns. ( D ) Representative wide-field images of mitochondrial polarization in live WT and Mfn2 KO (clone A4) cells as indicated by TMRM staining. ( E ) Representative confocal images of GFP-parkin recruitment to mitochondria as a function of time in WT or Mfn2 KO (clone A4) U2OS cells, transfected with GFP-parkin and treated with 20 μM CCCP. Red asterisks indicate cells in which GFP-parkin has fully translocated to mitochondria. Scale bar, 20 microns. ( F ) Quantification of parkin recruitment in cells from ( E ). Data points represent mean ± SEM, n = 3 replicates per condition, with > 100 cells counted per condition for each replicate. n.s., not significant; *, p

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Sequencing, CRISPR, Clone Assay, Imaging, Staining, Transfection

    Parkin recruitment kinetics in cells lacking both Mfns and other mitochondria-ER tethering factors. ( A ) Immunoblot analysis of whole-cell lysates from cells cultured in glucose or galactose transfected with control siRNA or siRNA targeting Mfn1 (‘siMfn1’), Mfn2 (‘siMfn2’) or both mitofusins (‘siMfn1 +2’). ( B ) Representative confocal images of GFP-parkin recruitment to mitochondria as a function of time in U2OS:GFP-parkin cells treated with 20 μM CCCP. Red asterisks indicate cells in which GFP-parkin has fully translocated to mitochondria. Scale bar, 20 microns. ( C ) Quantification of parkin recruitment in cells from ( B ). Data points represent mean ± SEM, n = 3 replicates cells per condition, with > 100 cells counted per condition for each replicate. ( D ) Parkin recruitment at one hour CCCP in cells from ( B ) arranged as a histogram. Bars represent mean ± SEM. n.s., not significant; *, p

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: Parkin recruitment kinetics in cells lacking both Mfns and other mitochondria-ER tethering factors. ( A ) Immunoblot analysis of whole-cell lysates from cells cultured in glucose or galactose transfected with control siRNA or siRNA targeting Mfn1 (‘siMfn1’), Mfn2 (‘siMfn2’) or both mitofusins (‘siMfn1 +2’). ( B ) Representative confocal images of GFP-parkin recruitment to mitochondria as a function of time in U2OS:GFP-parkin cells treated with 20 μM CCCP. Red asterisks indicate cells in which GFP-parkin has fully translocated to mitochondria. Scale bar, 20 microns. ( C ) Quantification of parkin recruitment in cells from ( B ). Data points represent mean ± SEM, n = 3 replicates cells per condition, with > 100 cells counted per condition for each replicate. ( D ) Parkin recruitment at one hour CCCP in cells from ( B ) arranged as a histogram. Bars represent mean ± SEM. n.s., not significant; *, p

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Cell Culture, Transfection

    LC/MS of immunoprecipitated Mfn2. ( A ) Base peak chromatograms indicating equal loading of both DMSO- and CCCP-treated samples from Figure 2F and G . ( B ) Extracted ion chromatograms of the indicated Ub and Mfn2 peptides from both DMSO- (blue line) and CCCP- (red line) treated samples.

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: LC/MS of immunoprecipitated Mfn2. ( A ) Base peak chromatograms indicating equal loading of both DMSO- and CCCP-treated samples from Figure 2F and G . ( B ) Extracted ion chromatograms of the indicated Ub and Mfn2 peptides from both DMSO- (blue line) and CCCP- (red line) treated samples.

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation

    Mfn2 is a mitochondrion-ER tether. ( A ) Representative TEM images of U2OS:GFP-parkin cells transfected with the indicated siRNA. ER tubules are pseudocoloured blue. Scale bar, 500 nm. ( B–D ) Quantification of mitochondrial length ( B ), relative percentage of OMM in contact with the ER ( C ) and percentage of mitochondria in contact with ER per field of view ( D ) in cells from ( A ). Bars represent mean ± SEM, n = 66 to 70 mitochondria in 5 to 7 fields per condition. n.s., not significant; *, p

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: Mfn2 is a mitochondrion-ER tether. ( A ) Representative TEM images of U2OS:GFP-parkin cells transfected with the indicated siRNA. ER tubules are pseudocoloured blue. Scale bar, 500 nm. ( B–D ) Quantification of mitochondrial length ( B ), relative percentage of OMM in contact with the ER ( C ) and percentage of mitochondria in contact with ER per field of view ( D ) in cells from ( A ). Bars represent mean ± SEM, n = 66 to 70 mitochondria in 5 to 7 fields per condition. n.s., not significant; *, p

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Transmission Electron Microscopy, Transfection

    Location and conservation of ubiquitination and phosphorylation sites in Mfn2. ( A ) Sequence alignment of sites of Mfn2 modification across species. Ubiquitinated lysines and phosphorylated serines and threonines are indicated by arrowheads. Residue numbering is according to the human sequence. HR, heptad repeat domain. ( B ) Diagram of Mfn2 post-translational modification by parkin-mediated ubiquitination ( Sarraf et al., 2013 ) and PINK1-mediated phosphorylation ( Chen and Dorn, 2013 ) for both double- (top) and single- (bottom) pass topologies. Phosphosites are denoted in red, while sites of ubiquitination are marked in grey. HR, heptad repeat domain; OMM, outer mitochondrial membrane.

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: Location and conservation of ubiquitination and phosphorylation sites in Mfn2. ( A ) Sequence alignment of sites of Mfn2 modification across species. Ubiquitinated lysines and phosphorylated serines and threonines are indicated by arrowheads. Residue numbering is according to the human sequence. HR, heptad repeat domain. ( B ) Diagram of Mfn2 post-translational modification by parkin-mediated ubiquitination ( Sarraf et al., 2013 ) and PINK1-mediated phosphorylation ( Chen and Dorn, 2013 ) for both double- (top) and single- (bottom) pass topologies. Phosphosites are denoted in red, while sites of ubiquitination are marked in grey. HR, heptad repeat domain; OMM, outer mitochondrial membrane.

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Sequencing, Modification

    p97 governs ER-OMM contact via the extraction of Mfn2 complexes. ( A ) Immunoblot analysis of NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP for the indicated time, separated by blue native- (BN-) and SDS-PAGE. ( B, C ) Immunoblot analysis of Mfn1- ( B ) and Mfn2- ( C ) containing complexes in NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT and C431S cells treated with 20 μM CCCP for four hours, separated by BN- and SDS-PAGE. ( D ) Mitochondria isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP for one hour were, after solubilization in NP-40, incubated with 1 μM Usp2 for 30 min at 37°C prior to separation by SDS-PAGE. Red asterisks indicate ubiquitinated species of Mfn1 and Mfn2. Densitometry calculations for the Mfn1 and Mfn2 bands (shorter exposure) relative to CIII-core2 are shown under the respective immunoblots. ( E ) Immunoblot analysis of NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP in the presence or absence of 25 μM NMS-873 for the indicated time, separated by blue native- (BN-) and SDS-PAGE. Red asterisks indicate ubiquinated Mfn species visible by SDS-PAGE, while the arrowhead denotes the unmodified band. ( F ) Representative TEM images of mitochondria in contact with ER (pseudocoloured blue) in U2OS:GFP-parkin cells treated with 20 μM CCCP (‘+CCCP’) for four hours in the presence or absence of 25 μM NMS-873. Scale bar, 500 nm. ( G,H ) Quantification of TEM from ( F ) in cells treated with (blue bars) or without (red bars) 20 μM CCCP for four hours. The percent of OMM per mitochondrion ( G ) and mitochondria per field ( H ) in contact with the ER was quantified. Bars represent mean ± SEM, n = 99 to 187 mitochondria in 12 to 14 fields per condition. n.s., not significant; *, p

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: p97 governs ER-OMM contact via the extraction of Mfn2 complexes. ( A ) Immunoblot analysis of NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP for the indicated time, separated by blue native- (BN-) and SDS-PAGE. ( B, C ) Immunoblot analysis of Mfn1- ( B ) and Mfn2- ( C ) containing complexes in NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT and C431S cells treated with 20 μM CCCP for four hours, separated by BN- and SDS-PAGE. ( D ) Mitochondria isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP for one hour were, after solubilization in NP-40, incubated with 1 μM Usp2 for 30 min at 37°C prior to separation by SDS-PAGE. Red asterisks indicate ubiquitinated species of Mfn1 and Mfn2. Densitometry calculations for the Mfn1 and Mfn2 bands (shorter exposure) relative to CIII-core2 are shown under the respective immunoblots. ( E ) Immunoblot analysis of NP-40-solubilized mitochondria, isolated from U2OS:GFP-parkin WT cells treated with 20 μM CCCP in the presence or absence of 25 μM NMS-873 for the indicated time, separated by blue native- (BN-) and SDS-PAGE. Red asterisks indicate ubiquinated Mfn species visible by SDS-PAGE, while the arrowhead denotes the unmodified band. ( F ) Representative TEM images of mitochondria in contact with ER (pseudocoloured blue) in U2OS:GFP-parkin cells treated with 20 μM CCCP (‘+CCCP’) for four hours in the presence or absence of 25 μM NMS-873. Scale bar, 500 nm. ( G,H ) Quantification of TEM from ( F ) in cells treated with (blue bars) or without (red bars) 20 μM CCCP for four hours. The percent of OMM per mitochondrion ( G ) and mitochondria per field ( H ) in contact with the ER was quantified. Bars represent mean ± SEM, n = 99 to 187 mitochondria in 12 to 14 fields per condition. n.s., not significant; *, p

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Isolation, SDS Page, Incubation, Western Blot, Transmission Electron Microscopy

    p97 and Mfn2 effect mitophagy through parkin substrate availability. ( A ) U2OS:mtKeima cells were transfected with the indicated siRNA and GFP-parkin WT, and were treated with 20 μM CCCP (or DMSO) for five hours in the presence (dark grey box) or absence (light grey box) of 25 μM NMS-873. mtKeima fluorescence in GFP-positive cells was measured using flow cytometry by excitation at 405 nm (neutral pH) and 561 nm (acidified). The data are represented as scatter plots of fluorescence emission from excitation at both wavelengths. The gated area encloses cells undergoing mitophagy and the percentage of cells within this gate is indicated in the top-left corner of each plot. ( B ) Quantification of the percent of cells undergoing mitophagy in cells from ( A ), expressed as a ratio of CCCP-treated cells to those treated with DMSO. Bars represent mean ± SEM, n = 2 experiments. n.s., not significant; ****, p

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: p97 and Mfn2 effect mitophagy through parkin substrate availability. ( A ) U2OS:mtKeima cells were transfected with the indicated siRNA and GFP-parkin WT, and were treated with 20 μM CCCP (or DMSO) for five hours in the presence (dark grey box) or absence (light grey box) of 25 μM NMS-873. mtKeima fluorescence in GFP-positive cells was measured using flow cytometry by excitation at 405 nm (neutral pH) and 561 nm (acidified). The data are represented as scatter plots of fluorescence emission from excitation at both wavelengths. The gated area encloses cells undergoing mitophagy and the percentage of cells within this gate is indicated in the top-left corner of each plot. ( B ) Quantification of the percent of cells undergoing mitophagy in cells from ( A ), expressed as a ratio of CCCP-treated cells to those treated with DMSO. Bars represent mean ± SEM, n = 2 experiments. n.s., not significant; ****, p

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Transfection, Fluorescence, Flow Cytometry, Cytometry

    Dismantling of Mfn2 interorganellar bridges by PINK1, parkin and p97 during mitophagy. ( A ) PINK1-phosphorylated Ub on Mfn2 initially recruits parkin to Mfn2 complexes, where it is phosphorylated and activated by PINK1. ( B ) Parkin and PINK1 cooperate to catalyze a pUb burst on Mfn2. ( C ) Ubiquitinated Mfn2 HMW complexes are recognized by p97, which translocates to mitochondria. ( D ) Ubiquitinated Mfn2 is retrotranslocated from the OMM and degraded by the proteasome. ( E ) VDACs and possibly other substrates become available to the parkin/PINK1 system, and their phosphoubiquitination stabilizes parkin on mitochondria to drive mitophagy.

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: Dismantling of Mfn2 interorganellar bridges by PINK1, parkin and p97 during mitophagy. ( A ) PINK1-phosphorylated Ub on Mfn2 initially recruits parkin to Mfn2 complexes, where it is phosphorylated and activated by PINK1. ( B ) Parkin and PINK1 cooperate to catalyze a pUb burst on Mfn2. ( C ) Ubiquitinated Mfn2 HMW complexes are recognized by p97, which translocates to mitochondria. ( D ) Ubiquitinated Mfn2 is retrotranslocated from the OMM and degraded by the proteasome. ( E ) VDACs and possibly other substrates become available to the parkin/PINK1 system, and their phosphoubiquitination stabilizes parkin on mitochondria to drive mitophagy.

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques:

    Parkin recruitment in Mfn2-depleted cells requires PINK1 and phosphoubiquitin binding. ( A ) Immunoblot analysis of PINK1 depletion in WT and Mfn2 KO (clone A4) U2OS cells treated with 20 μM CCCP for four hours. The arrowhead indicates the PINK1 band, while the asterisk indicates a non-specific band. ( B ) U2OS cells from ( A ) were transfected with GFP-parkin and treated with 20 μM CCCP for four hours prior to fixation. Red asterisks mark cells in which parkin has been recruited to mitochondria. Scale bar, 10 microns. ( C ) Quantification of parkin-expressing cells from ( A ), left untreated (red bars) or treated with 20 μM CCCP for four hours (blue bars). Bars represent mean ± SEM, n = 3 replicates cells per condition, with > 100 GFP-positive cells counted per condition for each replicate. n.s., not significant; ****, p

    Journal: eLife

    Article Title: Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy

    doi: 10.7554/eLife.32866

    Figure Lengend Snippet: Parkin recruitment in Mfn2-depleted cells requires PINK1 and phosphoubiquitin binding. ( A ) Immunoblot analysis of PINK1 depletion in WT and Mfn2 KO (clone A4) U2OS cells treated with 20 μM CCCP for four hours. The arrowhead indicates the PINK1 band, while the asterisk indicates a non-specific band. ( B ) U2OS cells from ( A ) were transfected with GFP-parkin and treated with 20 μM CCCP for four hours prior to fixation. Red asterisks mark cells in which parkin has been recruited to mitochondria. Scale bar, 10 microns. ( C ) Quantification of parkin-expressing cells from ( A ), left untreated (red bars) or treated with 20 μM CCCP for four hours (blue bars). Bars represent mean ± SEM, n = 3 replicates cells per condition, with > 100 GFP-positive cells counted per condition for each replicate. n.s., not significant; ****, p

    Article Snippet: Antibodies and other reagents Antibodies used in this study include anti-actin (Millipore, MAB1501), anti-β-III tubulin (Sigma-Aldrich, T8660), anti-Cardif (referred to herein as MAVS, Enzo Life Sciences, ALX-210–929 C100), anti-cytochrome c (BD Biosciences, 556432), anti-GFP (ab6673, Abcam), anti-GFP (A6455, Invitrogen), anti-Grp78 (Santa Cruz, sc-376768), anti-HA (Abcam, ab9134), anti-HK1 (Cell Signaling Technology, 2024S), anti-Mfn1 (Santa Cruz, sc-50330), anti-Mfn2 (Sigma-Aldrich, M6319), anti-Mfn2 (Cell Signaling, 9482), anti-MTCO1 (herein referred to as CIV-COXI, ab14705), anti-p62 (Progen, GP62-C), anti-PDH E1a (Abcam, ab110330), anti-PDH E2/E3bp (Abcam, ab110333), anti-PDI (Abcam, ab2792), anti-PINK1 (Cell Signaling, 6946), anti-pS65 ubiquitin (Millipore, ABS1513-I), anti-Rab11A (Cell Signaling, 2413), anti-Rhot1 (referred to herein as Miro1, Sigma-Aldrich, HPA010687), anti-SDHA (referred to herein as CII-SDHA, Abcam, ab14715), anti-Stx17 (ProteinTech, 17815–1-AP), anti-TH (Pel-Freez, P40101-150), anti-TIM23 (BD, 611222), anti-TOM20 (Santa Cruz, sc-11414), anti-TOM70 (Santa Cruz, sc-390545), anti-ubiquitin [FK2] (Enzo Life Sciences, BML-PW8810), anti-ubiquitin [P4DI] (Santa Cruz, sc-8017), anti-UQCRC2 (referred to herein as CIII-core2, Abcam, ab14745), anti-UQCRFS1 (referred to herein as CIII-Rieske, Abcam, ab14746), anti-VCP (referred to herein as p97, Abcam, ab11433) and anti-VDAC1 (Abcam, ab14734).

    Techniques: Binding Assay, Transfection, Expressing

    The expression of markers of mitochondrial fusion, fission and biogenesis is affected by tumor and drug-induced cachexia. (A) Representative western blotting for OPA-1, Mitofusin-2, DRP-1, Cytochrome-C (Cyt-C), and PGC-1α in the muscle of C26 hosts or mice exposed to Folfiri. (B,C) Quantification of the bands ( n = 4). Significance of the differences: * p

    Journal: Frontiers in Physiology

    Article Title: Cancer and Chemotherapy Contribute to Muscle Loss by Activating Common Signaling Pathways

    doi: 10.3389/fphys.2016.00472

    Figure Lengend Snippet: The expression of markers of mitochondrial fusion, fission and biogenesis is affected by tumor and drug-induced cachexia. (A) Representative western blotting for OPA-1, Mitofusin-2, DRP-1, Cytochrome-C (Cyt-C), and PGC-1α in the muscle of C26 hosts or mice exposed to Folfiri. (B,C) Quantification of the bands ( n = 4). Significance of the differences: * p

    Article Snippet: Antibodies used were OPA-1 (#80471), Mitofusin-2 (#9482), DRP-1 (#8570), Cytochrome C (#11940) from Cell Signaling Technologies (Danvers, MA), PGC-1α (#ab3242) from Abcam (Cambridge, MA) and α-Tubulin (#12G10) from Developmental Studies Hybridoma Bank (Iowa City, IA).

    Techniques: Expressing, Western Blot, Pyrolysis Gas Chromatography, Mouse Assay

    BRD9 is part of the SWI-SNF complex and binds MYC enhancer sequences. ( A ) Immunoblots showing Co-IP of BRD9 with the SWI-SNF subunit BRG1 from MCF-10A nuclear extracts or ( B ) nuclear extracts isolated from DKI cells transduced with lenti-BRD9. ( C ) ChIP data (Mean ± SD) showing BRD9 occupancy at MYC super-enhancer and VIM promoter DNA regions. BRD4 and SUMO2 represent positive control sequences; NEG represents a non-specific negative control sequence. ( D ) RT-qPCR (Mean ± SD) of MYC transcript in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. ( E ) Immunoblot data showing MYC (with densitometric quantification, lower panel), target NDRG1, and VIM expression in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. **, p

    Journal: Cancers

    Article Title: PIK3CA Cooperates with KRAS to Promote MYC Activity and Tumorigenesis via the Bromodomain Protein BRD9

    doi: 10.3390/cancers11111634

    Figure Lengend Snippet: BRD9 is part of the SWI-SNF complex and binds MYC enhancer sequences. ( A ) Immunoblots showing Co-IP of BRD9 with the SWI-SNF subunit BRG1 from MCF-10A nuclear extracts or ( B ) nuclear extracts isolated from DKI cells transduced with lenti-BRD9. ( C ) ChIP data (Mean ± SD) showing BRD9 occupancy at MYC super-enhancer and VIM promoter DNA regions. BRD4 and SUMO2 represent positive control sequences; NEG represents a non-specific negative control sequence. ( D ) RT-qPCR (Mean ± SD) of MYC transcript in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. ( E ) Immunoblot data showing MYC (with densitometric quantification, lower panel), target NDRG1, and VIM expression in DKI cells transduced with empty (control), or BRD9-encoding lentiviral constructs. **, p

    Article Snippet: Primary antibodies used in this study are: anti-RAS (#3965), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370S), anti-p44/42 MAPK (ERK1/2) (#4696), anti-PI3K p110α (#4255), anti-phospho-AKT (Ser473) (#4051), anti-AKT (#4685), anti-C-MYC (#9402), anti-NDRG1 (#5196), anti-GAPDH (#2118) (Cell Signaling Technologies, Danvers, MA, USA), anti-BRD9 (#A303-781A, Bethyl Laboratories, Montgomery, TX, USA), anti-BRG1 (#sc-17796) and anti-VIM (#sc-6260) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Western Blot, Co-Immunoprecipitation Assay, Isolation, Transduction, Chromatin Immunoprecipitation, Positive Control, Negative Control, Sequencing, Quantitative RT-PCR, Construct, Expressing

    Inhibition of MYC blocks anchorage-independent growth in double mutant cells. ( A ) RT-qPCR da ta (Mean ± SD) showing MYC expression in MCF-10A WT and mutant cells after 24 h incubation with 20 µM I-BRD9. ( B ) Protein levels of MYC and target NDRG1 as well as VIM in DKI cells after 24 h treatment with 20 µM I-BRD9. ( C ) Soft agar growth of DKI cells in the presence of the MYC inhibitor KJ-Pyr-9 for 15 days. ***, p

    Journal: Cancers

    Article Title: PIK3CA Cooperates with KRAS to Promote MYC Activity and Tumorigenesis via the Bromodomain Protein BRD9

    doi: 10.3390/cancers11111634

    Figure Lengend Snippet: Inhibition of MYC blocks anchorage-independent growth in double mutant cells. ( A ) RT-qPCR da ta (Mean ± SD) showing MYC expression in MCF-10A WT and mutant cells after 24 h incubation with 20 µM I-BRD9. ( B ) Protein levels of MYC and target NDRG1 as well as VIM in DKI cells after 24 h treatment with 20 µM I-BRD9. ( C ) Soft agar growth of DKI cells in the presence of the MYC inhibitor KJ-Pyr-9 for 15 days. ***, p

    Article Snippet: Primary antibodies used in this study are: anti-RAS (#3965), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4370S), anti-p44/42 MAPK (ERK1/2) (#4696), anti-PI3K p110α (#4255), anti-phospho-AKT (Ser473) (#4051), anti-AKT (#4685), anti-C-MYC (#9402), anti-NDRG1 (#5196), anti-GAPDH (#2118) (Cell Signaling Technologies, Danvers, MA, USA), anti-BRD9 (#A303-781A, Bethyl Laboratories, Montgomery, TX, USA), anti-BRG1 (#sc-17796) and anti-VIM (#sc-6260) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Inhibition, Mutagenesis, Quantitative RT-PCR, Expressing, Incubation

    Loss of SOX4 significantly inhibits PTEN deletion-induced prostate tumorigenesis. ( A ) Representative images of prostate glands from wild-type, Pten −/− and Pten −/− /Sox4 −/− mice are shown. Homozygous deletion

    Journal: Cancer research

    Article Title: SOX4 is essential for prostate tumorigenesis initiated by PTEN ablation

    doi: 10.1158/0008-5472.CAN-15-1868

    Figure Lengend Snippet: Loss of SOX4 significantly inhibits PTEN deletion-induced prostate tumorigenesis. ( A ) Representative images of prostate glands from wild-type, Pten −/− and Pten −/− /Sox4 −/− mice are shown. Homozygous deletion

    Article Snippet: Consistent with the essential role of SOX4 in stimulation of cell proliferation, our results indicate that loss of SOX4 significantly reduces prostate cancer cell proliferation in vivo .

    Techniques: Mouse Assay

    Schematic diagram of a proposed model illustrating how SOX4 interacts with PI3K-AKT, Wnt-β-catenin, and AR signaling pathways, and how the PI3K-AKT-mTOR pathway regulates the expression of SOX4 in prostate cancer progression. A potential positive

    Journal: Cancer research

    Article Title: SOX4 is essential for prostate tumorigenesis initiated by PTEN ablation

    doi: 10.1158/0008-5472.CAN-15-1868

    Figure Lengend Snippet: Schematic diagram of a proposed model illustrating how SOX4 interacts with PI3K-AKT, Wnt-β-catenin, and AR signaling pathways, and how the PI3K-AKT-mTOR pathway regulates the expression of SOX4 in prostate cancer progression. A potential positive

    Article Snippet: Consistent with the essential role of SOX4 in stimulation of cell proliferation, our results indicate that loss of SOX4 significantly reduces prostate cancer cell proliferation in vivo .

    Techniques: Expressing

    Generation of prostate epithelium-specific Sox4 and Pten conditional double-knockout mice. ( A ) Schematic representation of the experimental setup for tamoxifen treatment. Mice at eight weeks of age were injected with tamoxifen (1 mg/kg) or vehicle i.p.

    Journal: Cancer research

    Article Title: SOX4 is essential for prostate tumorigenesis initiated by PTEN ablation

    doi: 10.1158/0008-5472.CAN-15-1868

    Figure Lengend Snippet: Generation of prostate epithelium-specific Sox4 and Pten conditional double-knockout mice. ( A ) Schematic representation of the experimental setup for tamoxifen treatment. Mice at eight weeks of age were injected with tamoxifen (1 mg/kg) or vehicle i.p.

    Article Snippet: Consistent with the essential role of SOX4 in stimulation of cell proliferation, our results indicate that loss of SOX4 significantly reduces prostate cancer cell proliferation in vivo .

    Techniques: Double Knockout, Mouse Assay, Injection

    Loss of SOX4 inhibits activation of AKT and β-catenin. ( A ) Representative images of immunohistochemical staining of phospho-AKT (Ser473) and active β-catenin in the dorsolateral prostate lobes of wild-type, Sox4 −/− , Pten

    Journal: Cancer research

    Article Title: SOX4 is essential for prostate tumorigenesis initiated by PTEN ablation

    doi: 10.1158/0008-5472.CAN-15-1868

    Figure Lengend Snippet: Loss of SOX4 inhibits activation of AKT and β-catenin. ( A ) Representative images of immunohistochemical staining of phospho-AKT (Ser473) and active β-catenin in the dorsolateral prostate lobes of wild-type, Sox4 −/− , Pten

    Article Snippet: Consistent with the essential role of SOX4 in stimulation of cell proliferation, our results indicate that loss of SOX4 significantly reduces prostate cancer cell proliferation in vivo .

    Techniques: Activation Assay, Immunohistochemistry, Staining

    SOX4 expression is regulated by the PI3K-AKT-mTOR signaling pathway. ( A–C ) Human prostate cancer cell line LNCaP was serum-starved for 24 hours, followed by the treatment with 50 µM of PI3K inhibitor LY294002 ( A ) or 2 µM of AKT

    Journal: Cancer research

    Article Title: SOX4 is essential for prostate tumorigenesis initiated by PTEN ablation

    doi: 10.1158/0008-5472.CAN-15-1868

    Figure Lengend Snippet: SOX4 expression is regulated by the PI3K-AKT-mTOR signaling pathway. ( A–C ) Human prostate cancer cell line LNCaP was serum-starved for 24 hours, followed by the treatment with 50 µM of PI3K inhibitor LY294002 ( A ) or 2 µM of AKT

    Article Snippet: Consistent with the essential role of SOX4 in stimulation of cell proliferation, our results indicate that loss of SOX4 significantly reduces prostate cancer cell proliferation in vivo .

    Techniques: Expressing

    CYP6D1v1 promoter sequence from −365 to −267 (from LPR) is able to mediate PB induction. Promoter constructs are numbered relative to the transcription start site (TSS) at +1. Relative luciferase activity was estimated by normalizing each signal of each promoter construct to the mean of signals of pGL3-Basic vector in the same replicate. Bars represent the mean relative luciferase activity ± S.D. of three independent transfections. Gray bars represent the signal in the presence of PB and white bars represent the control. Double asterisks indicate a greater PB induction relative to the next shorter promoter construct (p

    Journal: Pesticide biochemistry and physiology

    Article Title: Investigations of the constitutive overexpression of CYP6D1 in the permethrin resistantLPR strain of house fly (Musca domestica)

    doi: 10.1016/j.pestbp.2011.02.012

    Figure Lengend Snippet: CYP6D1v1 promoter sequence from −365 to −267 (from LPR) is able to mediate PB induction. Promoter constructs are numbered relative to the transcription start site (TSS) at +1. Relative luciferase activity was estimated by normalizing each signal of each promoter construct to the mean of signals of pGL3-Basic vector in the same replicate. Bars represent the mean relative luciferase activity ± S.D. of three independent transfections. Gray bars represent the signal in the presence of PB and white bars represent the control. Double asterisks indicate a greater PB induction relative to the next shorter promoter construct (p

    Article Snippet: Promoter regions −925/+85, −365/+85, −267/+85, and −57/+85 (numbers are relative to transcription start site, defined as +1) were constructed into restriction enzyme sites Sac I and Bgl II of pGL3-Basic vector (Promega, Madison, WI) as previously described [ ].

    Techniques: Sequencing, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection

    Detection of the sites activated by E2F. ( A ) Three regions of the p68 gene promoter (+9/+16, –11/–3 and –19/–12) were mutated (the mutations were transversions of each base in the three base stretch and are indicated by X). These sites are represented by hatched boxes. Each mutant plasmid was co-transfected into NIH 3T3 cells with a plasmid carrying CMV-E2F-1 or CMV. The promoter activities are indicated as relative activity with respect to the activity of pKL12 without E2F-1. The promoter activity with and without E2F-1 is represented by open and closed bars, respectively. The increase in the relative activity of luciferase with CMV-E2F-1 is presented on the right. ( B ) The wild-type sequence and mutation sequence. Open boxes indicate three homologous E2F-binding sites and arrows represent transversion mutations.

    Journal: Nucleic Acids Research

    Article Title: Cloning and characterization of the 5?-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase ? 68 kDa subunit gene

    doi:

    Figure Lengend Snippet: Detection of the sites activated by E2F. ( A ) Three regions of the p68 gene promoter (+9/+16, –11/–3 and –19/–12) were mutated (the mutations were transversions of each base in the three base stretch and are indicated by X). These sites are represented by hatched boxes. Each mutant plasmid was co-transfected into NIH 3T3 cells with a plasmid carrying CMV-E2F-1 or CMV. The promoter activities are indicated as relative activity with respect to the activity of pKL12 without E2F-1. The promoter activity with and without E2F-1 is represented by open and closed bars, respectively. The increase in the relative activity of luciferase with CMV-E2F-1 is presented on the right. ( B ) The wild-type sequence and mutation sequence. Open boxes indicate three homologous E2F-binding sites and arrows represent transversion mutations.

    Article Snippet: The 5′-end deletion mutants, pS12(–547) and pKL12(–164), derived from p6E(–995), were created using exonuclease III (TaKaRa Shuzou, Japan).

    Techniques: Mutagenesis, Plasmid Preparation, Transfection, Activity Assay, Luciferase, Sequencing, Binding Assay

    Activation of mTOR and increased TGFβ1 ligand in murine SGTs. (A) Immunohistochemistry staining of p-mTOR (brown) in SPAs developed from Pten SG-KO mice ( n = 7) or Smad4 SG-KO mice ( n = 5), and SACCs developed from Pten.Smad4 SG-KO mice ( n = 6). WT: wild-type. Scale bar: 50 μm. (B) ELISA measurement of TGFβ1 levels in SPAs developed from Pten SG-KO mice or Smad4 SG-KO mice, and SACCs developed from Pten.Smad4 SG-KO mice. n = 3 of each group. * P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression

    doi: 10.1016/j.neo.2018.05.009

    Figure Lengend Snippet: Activation of mTOR and increased TGFβ1 ligand in murine SGTs. (A) Immunohistochemistry staining of p-mTOR (brown) in SPAs developed from Pten SG-KO mice ( n = 7) or Smad4 SG-KO mice ( n = 5), and SACCs developed from Pten.Smad4 SG-KO mice ( n = 6). WT: wild-type. Scale bar: 50 μm. (B) ELISA measurement of TGFβ1 levels in SPAs developed from Pten SG-KO mice or Smad4 SG-KO mice, and SACCs developed from Pten.Smad4 SG-KO mice. n = 3 of each group. * P

    Article Snippet: This synergistic suppression effect of Pten and Smad4 on tumor progression has also been observed in other cancer types, such as pancreas , forestomach , and liver .

    Techniques: Activation Assay, Immunohistochemistry, Staining, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Combined deletion of Pten and Smad4 in murine salivary glands caused malignant SGTs. (A) Gross picture of a salivary gland tumor (dotted circle) developed in a Pten.Smad4 SG-KO mouse. (B) H E staining of a murine SACC developed in a Pten.Smad4 SG-KO mouse. Arrow showed the “Swiss cheese” change, which is a special pathological characteristic found in human SACCs. Scale bar: 100 μm. (C) Representative immunohistochemistry staining of keratin 5 (brown) in the murine SACCs ( n = 6). Scale bar: 50 μm. (D) Representative immunohistochemistry staining of smooth muscle actin (brown) in the murine SACCs ( n = 6). Scale bar: 50 μm. (E) Representative immunohistochemistry staining of p63 (brown) in the murine SACCs ( n = 6). Scale bar: 50 μm. (F) Representative immunohistochemistry staining of Ki67 (brown) in the murine SACCs ( n = 6). Scale bar: 25 μm.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression

    doi: 10.1016/j.neo.2018.05.009

    Figure Lengend Snippet: Combined deletion of Pten and Smad4 in murine salivary glands caused malignant SGTs. (A) Gross picture of a salivary gland tumor (dotted circle) developed in a Pten.Smad4 SG-KO mouse. (B) H E staining of a murine SACC developed in a Pten.Smad4 SG-KO mouse. Arrow showed the “Swiss cheese” change, which is a special pathological characteristic found in human SACCs. Scale bar: 100 μm. (C) Representative immunohistochemistry staining of keratin 5 (brown) in the murine SACCs ( n = 6). Scale bar: 50 μm. (D) Representative immunohistochemistry staining of smooth muscle actin (brown) in the murine SACCs ( n = 6). Scale bar: 50 μm. (E) Representative immunohistochemistry staining of p63 (brown) in the murine SACCs ( n = 6). Scale bar: 50 μm. (F) Representative immunohistochemistry staining of Ki67 (brown) in the murine SACCs ( n = 6). Scale bar: 25 μm.

    Article Snippet: This synergistic suppression effect of Pten and Smad4 on tumor progression has also been observed in other cancer types, such as pancreas , forestomach , and liver .

    Techniques: Staining, Immunohistochemistry

    Development of SGTs in the Pten HN-KO or the Smad4 HN-KO mice. (A) Representative gross pictures of salivary gland tumors (dotted circles) developed in head and neck–specific Pten knockout mice (left, Pten HN-KO , n = 5) or Smad4 knockout mice (right, Smad4 HN-KO , n = 3). (B) Representative H E staining at lower magnification of salivary pleomorphic adenomas developed in salivary glands of Pten HN-KO mice (left, n = 5) or Smad4 HN-KO mice (right, n = 3). Scale bar: 200 μm. (C) Representative HE staining at higher magnification of salivary pleomorphic adenomas developed in salivary glands of Pten HN-KO mice (left, n = 5) or Smad4 HN-KO mice (right, n = 3). Scale bar: 100 μm. (D) Representative immunohistochemistry staining of smooth muscle actin (brown) in murine salivary pleomorphic adenomas of Pten HN-KO mice (left, n = 5) or Smad4 HN-KO mice (right, n = 3). Scale bar: 50 μm.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression

    doi: 10.1016/j.neo.2018.05.009

    Figure Lengend Snippet: Development of SGTs in the Pten HN-KO or the Smad4 HN-KO mice. (A) Representative gross pictures of salivary gland tumors (dotted circles) developed in head and neck–specific Pten knockout mice (left, Pten HN-KO , n = 5) or Smad4 knockout mice (right, Smad4 HN-KO , n = 3). (B) Representative H E staining at lower magnification of salivary pleomorphic adenomas developed in salivary glands of Pten HN-KO mice (left, n = 5) or Smad4 HN-KO mice (right, n = 3). Scale bar: 200 μm. (C) Representative HE staining at higher magnification of salivary pleomorphic adenomas developed in salivary glands of Pten HN-KO mice (left, n = 5) or Smad4 HN-KO mice (right, n = 3). Scale bar: 100 μm. (D) Representative immunohistochemistry staining of smooth muscle actin (brown) in murine salivary pleomorphic adenomas of Pten HN-KO mice (left, n = 5) or Smad4 HN-KO mice (right, n = 3). Scale bar: 50 μm.

    Article Snippet: This synergistic suppression effect of Pten and Smad4 on tumor progression has also been observed in other cancer types, such as pancreas , forestomach , and liver .

    Techniques: Mouse Assay, Knock-Out, Staining, Immunohistochemistry

    Reduced expression of Pten and/or Smad4 in human salivary gland tumors. (A) Double IF staining of Pten (green) and Smad4 (red) in normal human salivary glands. Double positive staining of Pten and Smad4 was seen in the ductal cells (yellow); Smad4-positive staining was seen in seromucous cells (red). Scale bar: 50 μm. (B) Double IF staining of Pten (green) and Smad4 (red) in human SPAs. Examples of double positive staining (yellow) of Pten and Smad4 (Pten+/Smad4+) is shown in the left panel, Pten-negative staining alone (red, Pten−/Smad4+) is shown in the middle panel, and Smad4-negative staining alone (green, Pten+/Smad4−) is shown in the right panel. Scale bar: 50 μm. (C) Double IF staining of Pten (green) and Smad4 (red) in human SACCs. Examples of double positive staining (yellow) of Pten and Smad4 (Pten+/Smad4+) is shown in the first panel, Pten-negative staining alone (red, Pten−/Smad4+) is shown in the second panel, Smad4-negative staining alone (green, Pten+/Smad4−) is shown in the third panel, and double negative staining (blue, Pten−/Smad4−) is shown in the fourth panel. Scale bar: 50 μm. (D) Pie chart of summary results of Pten and Smad4 double IF staining in human SPAs and SACCs. Double IF results from three forms of SACC, i.e., tubular, cribriform, and solid, are further shown in individual pie chart. Positive cases from each category are shown as percentage along each pie chart. * P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression

    doi: 10.1016/j.neo.2018.05.009

    Figure Lengend Snippet: Reduced expression of Pten and/or Smad4 in human salivary gland tumors. (A) Double IF staining of Pten (green) and Smad4 (red) in normal human salivary glands. Double positive staining of Pten and Smad4 was seen in the ductal cells (yellow); Smad4-positive staining was seen in seromucous cells (red). Scale bar: 50 μm. (B) Double IF staining of Pten (green) and Smad4 (red) in human SPAs. Examples of double positive staining (yellow) of Pten and Smad4 (Pten+/Smad4+) is shown in the left panel, Pten-negative staining alone (red, Pten−/Smad4+) is shown in the middle panel, and Smad4-negative staining alone (green, Pten+/Smad4−) is shown in the right panel. Scale bar: 50 μm. (C) Double IF staining of Pten (green) and Smad4 (red) in human SACCs. Examples of double positive staining (yellow) of Pten and Smad4 (Pten+/Smad4+) is shown in the first panel, Pten-negative staining alone (red, Pten−/Smad4+) is shown in the second panel, Smad4-negative staining alone (green, Pten+/Smad4−) is shown in the third panel, and double negative staining (blue, Pten−/Smad4−) is shown in the fourth panel. Scale bar: 50 μm. (D) Pie chart of summary results of Pten and Smad4 double IF staining in human SPAs and SACCs. Double IF results from three forms of SACC, i.e., tubular, cribriform, and solid, are further shown in individual pie chart. Positive cases from each category are shown as percentage along each pie chart. * P

    Article Snippet: This synergistic suppression effect of Pten and Smad4 on tumor progression has also been observed in other cancer types, such as pancreas , forestomach , and liver .

    Techniques: Expressing, Staining, Negative Staining

    Deletion of Pten or Smad4 alone in murine salivary gland developed salivary pleomorphic adenoma. (A) qRT-PCR measurement of Pten or Smad4 mRNA levels in salivary glands of either the K5CrePR1.Ptenf/f mice or the K5CrePR1.Smad4f/f mice treated by vehicle or RU486, n = 3 of each. * P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Cooperation Between Pten and Smad4 in Murine Salivary Gland Tumor Formation and Progression

    doi: 10.1016/j.neo.2018.05.009

    Figure Lengend Snippet: Deletion of Pten or Smad4 alone in murine salivary gland developed salivary pleomorphic adenoma. (A) qRT-PCR measurement of Pten or Smad4 mRNA levels in salivary glands of either the K5CrePR1.Ptenf/f mice or the K5CrePR1.Smad4f/f mice treated by vehicle or RU486, n = 3 of each. * P

    Article Snippet: This synergistic suppression effect of Pten and Smad4 on tumor progression has also been observed in other cancer types, such as pancreas , forestomach , and liver .

    Techniques: Quantitative RT-PCR, Mouse Assay

    Twist1 deletion in basal keratinocytes suppresses TPA-induced epidermal proliferation

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 deletion in basal keratinocytes suppresses TPA-induced epidermal proliferation

    Article Snippet: Very recently, using a similar mouse model of keratinocyte specific deletion of Twist1, Beck et al. reported that Twist1 was required for the development of skin tumors induced by the two-stage skin carcinogenesis protocol in both a p53-dependent and p53-independent manner ( ).

    Techniques:

    Twist1 expression regulates p53 localization and p21 transcription in the epidermis

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 expression regulates p53 localization and p21 transcription in the epidermis

    Article Snippet: Very recently, using a similar mouse model of keratinocyte specific deletion of Twist1, Beck et al. reported that Twist1 was required for the development of skin tumors induced by the two-stage skin carcinogenesis protocol in both a p53-dependent and p53-independent manner ( ).

    Techniques: Expressing

    Twist1 is a novel regulator of the cell cycle in epidermal keratinocytes

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 is a novel regulator of the cell cycle in epidermal keratinocytes

    Article Snippet: Very recently, using a similar mouse model of keratinocyte specific deletion of Twist1, Beck et al. reported that Twist1 was required for the development of skin tumors induced by the two-stage skin carcinogenesis protocol in both a p53-dependent and p53-independent manner ( ).

    Techniques:

    Induction of skin tumors in Twist1 KO mice in response to initiation with 7,12-dimenthylbenz[a]anthracene (DMBA) and promotion with 12- O -tetradecanoylphorbol-13-acetate (TPA)

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Induction of skin tumors in Twist1 KO mice in response to initiation with 7,12-dimenthylbenz[a]anthracene (DMBA) and promotion with 12- O -tetradecanoylphorbol-13-acetate (TPA)

    Article Snippet: Very recently, using a similar mouse model of keratinocyte specific deletion of Twist1, Beck et al. reported that Twist1 was required for the development of skin tumors induced by the two-stage skin carcinogenesis protocol in both a p53-dependent and p53-independent manner ( ).

    Techniques: Mouse Assay

    Twist1 deficiency in epidermal keratinocytes hinders proliferative capacity of the bulge region stem cell compartment

    Journal: Molecular carcinogenesis

    Article Title: Twist1 Regulates Keratinocyte Proliferation and Skin Tumor Promotion

    doi: 10.1002/mc.22335

    Figure Lengend Snippet: Twist1 deficiency in epidermal keratinocytes hinders proliferative capacity of the bulge region stem cell compartment

    Article Snippet: Very recently, using a similar mouse model of keratinocyte specific deletion of Twist1, Beck et al. reported that Twist1 was required for the development of skin tumors induced by the two-stage skin carcinogenesis protocol in both a p53-dependent and p53-independent manner ( ).

    Techniques:

    Functional characterization of CDKI variants. a Protein expression in whole cell lysates of HEK293 cells transfected with wild type or mutagenized cDNA expression constructs. Top panel was probed with the indicated specific anti-CDKI antibody, mid dle panel with anti-NPTII antibody (to assess transfection efficiency), and lower panel with anti-tubulin antibody (to assess protein loading). Relative level of wild-type (wt) and mutant protein expression is shown ( n =3), with wt protein level=1. b Protein level in nuclear and cytoplasmic extracts of HEK293 cells transfected with wild-type or mutagenized cDNA expression constructs. Top panels of cytoplasmic extract (CE), and bottom panels of nuclear extract (NE) were probed with the indicated specific anti-CDKI antibody, anti-tubulin (to assess protein loading in CE, and to assess the efficiency of sub-cellular fractionation in NE), or with anti-p84 (to assess protein loading in the NE, and to assess the efficiency of sub-cellular fractionation in the CE). Relative level of wt and mutant protein expression in CE and NE is shown ( n =3), with wt protein level=1, * p =0.04. c In vitro translated (IVT) proteins used for GST pull-down assay in ‘ D ’. 35 S-Methionine labeled wild-type or mutagenized proteins were generated by in vitro translation from cDNA expression constructs. IVT proteins were detected by SDS-PAGE and autoradiography. d Protein-binding to CDK2 or CDK6. GST pull-down assay for binding to GST, GST-CDK2, or GST-CDK6 of 35 S-methionine labeled IVT wt or mutagenized proteins. The bound IVT protein was detected by SDS-PAGE and autoradiography. Input lane contained 10 % of the IVT product incubated with GST, GST-CDK2, or GST-CDK6. Relative binding of wt and mutant protein to GST-fusion proteins is shown with wt protein binding= 1 ( n =3 for p15 and p18; n =2 for p21), ** p

    Journal: Hormones & cancer

    Article Title: Germline and Somatic Mutations in Cyclin-Dependent Kinase Inhibitor Genes CDKN1A, CDKN2B, and CDKN2C in Sporadic Parathyroid Adenomas

    doi: 10.1007/s12672-013-0147-9

    Figure Lengend Snippet: Functional characterization of CDKI variants. a Protein expression in whole cell lysates of HEK293 cells transfected with wild type or mutagenized cDNA expression constructs. Top panel was probed with the indicated specific anti-CDKI antibody, mid dle panel with anti-NPTII antibody (to assess transfection efficiency), and lower panel with anti-tubulin antibody (to assess protein loading). Relative level of wild-type (wt) and mutant protein expression is shown ( n =3), with wt protein level=1. b Protein level in nuclear and cytoplasmic extracts of HEK293 cells transfected with wild-type or mutagenized cDNA expression constructs. Top panels of cytoplasmic extract (CE), and bottom panels of nuclear extract (NE) were probed with the indicated specific anti-CDKI antibody, anti-tubulin (to assess protein loading in CE, and to assess the efficiency of sub-cellular fractionation in NE), or with anti-p84 (to assess protein loading in the NE, and to assess the efficiency of sub-cellular fractionation in the CE). Relative level of wt and mutant protein expression in CE and NE is shown ( n =3), with wt protein level=1, * p =0.04. c In vitro translated (IVT) proteins used for GST pull-down assay in ‘ D ’. 35 S-Methionine labeled wild-type or mutagenized proteins were generated by in vitro translation from cDNA expression constructs. IVT proteins were detected by SDS-PAGE and autoradiography. d Protein-binding to CDK2 or CDK6. GST pull-down assay for binding to GST, GST-CDK2, or GST-CDK6 of 35 S-methionine labeled IVT wt or mutagenized proteins. The bound IVT protein was detected by SDS-PAGE and autoradiography. Input lane contained 10 % of the IVT product incubated with GST, GST-CDK2, or GST-CDK6. Relative binding of wt and mutant protein to GST-fusion proteins is shown with wt protein binding= 1 ( n =3 for p15 and p18; n =2 for p21), ** p

    Article Snippet: Antibodies used were as follows: p15 (Cell Signaling, 4822), p18 (Santa Cruz, sc-1208), p21 (Santa Cruz, sc-6246), p84 (GeneTex, GTX70220), tubulin (Calbiochem, CP06), and NPT-II (Upstate, 06–747).

    Techniques: Functional Assay, Expressing, Transfection, Construct, Mutagenesis, Cell Fractionation, In Vitro, Pull Down Assay, Labeling, Generated, SDS Page, Autoradiography, Protein Binding, Binding Assay, Incubation

    p-NDRG1 is able to predict the outcome of EGFR -mutant NSCLC patients. (A and B) GSEA revealed an enrichment of glycolytic genes in patients with EGFR -mutant NSCLC tumors with high p-NDRG1 expression (A) and an enrichment of oxidative phosphorylation genes in EGFR -mutant NSCLC patients with low p-NDRG1 expression (B). NES, normalized enrichment score. FDR, false discovery rate. (C) Correlation between p-NDRG1 expression and overall survival (OS) in NSCLC patients with EGFR driver mutations. The reverse phase protein array (RPPA) data for the relevant patients were downloaded from The Cancer Proteome Atlas (TCGA) database. The X-axis shows batch-normalized protein expression levels of p-NDRG1. The survival time of each patient is shown in the Y-axis in months. Black line denotes the linear fit.

    Journal: Cancer letters

    Article Title: mTORC2 contributes to the metabolic reprogramming in EGFR tyrosine-kinase inhibitor resistant cells in non-small cell lung cancer

    doi: 10.1016/j.canlet.2018.07.025

    Figure Lengend Snippet: p-NDRG1 is able to predict the outcome of EGFR -mutant NSCLC patients. (A and B) GSEA revealed an enrichment of glycolytic genes in patients with EGFR -mutant NSCLC tumors with high p-NDRG1 expression (A) and an enrichment of oxidative phosphorylation genes in EGFR -mutant NSCLC patients with low p-NDRG1 expression (B). NES, normalized enrichment score. FDR, false discovery rate. (C) Correlation between p-NDRG1 expression and overall survival (OS) in NSCLC patients with EGFR driver mutations. The reverse phase protein array (RPPA) data for the relevant patients were downloaded from The Cancer Proteome Atlas (TCGA) database. The X-axis shows batch-normalized protein expression levels of p-NDRG1. The survival time of each patient is shown in the Y-axis in months. Black line denotes the linear fit.

    Article Snippet: The Rictor (#2114), phospho-tyrosine (#5465), phospho-NDRG1 (T346; #5482), NDRG1 (#9485), phospho-Akt (S473; #4060), Akt (#9272), phospho-S6 (S240/4; #2215), S6 (#2217), phospho-4EBPl (T37/46; #2855), 4EBP1 (#9452), phospho-EGFR (Y1068; #3777), EGFR (#4267) and MET (#8198) antibodies were from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Mutagenesis, Expressing, Protein Array

    Inhibition of mTORC2 signaling increases spare respiratory capacity in erlotinib-resistant cells. A) Whole-cell lysates from isogenic NSCLC cell lines were subjected to western blotting analysis of mTORC1 and mTORC2 activities. Values below the figures, relative changes normalized to isogenic erlotinib-sensitive cells. (B) Whole-cell lysates from PC9 cells and PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were subjected to western blotting analysis of Rictor, p-Akt, p-NDRG1 and β-actin. Values below the figures, relative changes normalized to PC9 shCon. (C) The relative spare repository capacity (SRC) of PC9 cells and PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were measured by Seahorse mito stress assay. (D) PC9 cells and PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were cultured in the presence or absence of glucose. The relative doubling time was calculated as described in Fig. 1 . (E) PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were treated with complete or glucose-free medium for 24 h. Whole-cell lysates were subjected to western blotting analysis. Values below the figures, relative changes normalized to PC9R shCon in glucose repletion condition. Bars, SEM. ***P

    Journal: Cancer letters

    Article Title: mTORC2 contributes to the metabolic reprogramming in EGFR tyrosine-kinase inhibitor resistant cells in non-small cell lung cancer

    doi: 10.1016/j.canlet.2018.07.025

    Figure Lengend Snippet: Inhibition of mTORC2 signaling increases spare respiratory capacity in erlotinib-resistant cells. A) Whole-cell lysates from isogenic NSCLC cell lines were subjected to western blotting analysis of mTORC1 and mTORC2 activities. Values below the figures, relative changes normalized to isogenic erlotinib-sensitive cells. (B) Whole-cell lysates from PC9 cells and PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were subjected to western blotting analysis of Rictor, p-Akt, p-NDRG1 and β-actin. Values below the figures, relative changes normalized to PC9 shCon. (C) The relative spare repository capacity (SRC) of PC9 cells and PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were measured by Seahorse mito stress assay. (D) PC9 cells and PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were cultured in the presence or absence of glucose. The relative doubling time was calculated as described in Fig. 1 . (E) PC9R cells stably transduced with the control shRNA (shCon) or Rictor shRNA (shRictor) were treated with complete or glucose-free medium for 24 h. Whole-cell lysates were subjected to western blotting analysis. Values below the figures, relative changes normalized to PC9R shCon in glucose repletion condition. Bars, SEM. ***P

    Article Snippet: The Rictor (#2114), phospho-tyrosine (#5465), phospho-NDRG1 (T346; #5482), NDRG1 (#9485), phospho-Akt (S473; #4060), Akt (#9272), phospho-S6 (S240/4; #2215), S6 (#2217), phospho-4EBPl (T37/46; #2855), 4EBP1 (#9452), phospho-EGFR (Y1068; #3777), EGFR (#4267) and MET (#8198) antibodies were from Cell Signaling Technologies (Danvers, MA, USA).

    Techniques: Inhibition, Western Blot, Stable Transfection, Transduction, shRNA, Cell Culture