Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Upregulation of the transcription factors POU3F2 and NTF3 during neuronal differentiation of NT2D1. a The protocol for neuronal induction of NT2D1 cells is schematized. b β3-tubulin staining for neuronal cells in NT2D1 cells untreated (non) and treated with neuronal induction medium at the indicated time points. c Quantification of β3-tubulin-positive cells. d Immunoblotting analysis for POU3F2, POU3F3, β3-tubulin, and NTF3 in NT2D1 cells untreated or treated with neuronal induction medium at the indicated time points. The values show the expression relative to that of untreated cells (to which a value of 1 was assigned). e Microarray analysis showed that neuronal induction for 6 h increased the expression of NTF3 and GADD45 in NT2D1 cells. f NTF3 mRNA expression after neuronal induction was analyzed by real-time PCR. The levels of mRNA were calculated as the relative expression compared with that of non-induced NT2D1 cells. GAPDH mRNA was used as a control. * p < 0.05; *** p < 0.001. g Phospho-TrkC (Tyr820) staining in treated and untreated NT2D1 cells. Values are presented as mean ± SEM of three independent experiments for c and f

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

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

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Identification of the POU3F2 binding site on the NTF3 promoter. a Transcription factor response elements predicted by the Transcription Element Search System for the nucleotide sequence of the NTF3 promoter region (− 1823 to + 243). The transcription start site is indicated as + 1. b Comparison of NTF3 promoter sequence conservation between different species. c Biotin-labeled oligonucleotides containing the intact or mutated POU3F2 binding site were hybridized with total lysates prepared from NT2D1 cells. The POU3F2-DNA complexes were precipitated by streptavidin agarose beads. POU3F2 was analyzed by Western blot analyses. The input of nuclear extracts was used as loading control. Three independent experiments were performed. d Chromatin was prepared from NT2D1 cells treated with induction medium for 0, 2, and 6 h. Cell lysates were mixed with antibodies against POU3F2 or IgG and then precipitated. The precipitates were analyzed by PCR for the presence of the NTF3 promoter sequence. The DNA purified from the sonicated chromatin was directly analyzed by PCR using the ChIP primer, which was used as an input control (Input). e The values of the ChIP DNA were normalized to that of the NT2D1 cells at 0 h (as a control). Values of fold-change over the control are presented as mean ± SEM of three independent experiments for d . * p < 0.05 compared with the control

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Binding Assay, Sequencing, Comparison, Labeling, Western Blot, Control, Purification, Sonication

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 on NTF3 promoter activity. a Schematic representation of NTF3-luciferase chimeric constructs. The negative numbers refer to the numbers of bases upstream of the transcription start (+ 1) site of the NTF3 gene. b NT2D1 cells were transiently transfected with the pGL3 basic vector or NTF3 promoter constructs of different lengths. The luciferase activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. c NT2D1 cells were transfected with the pGL3 basic vector, pNTF3-1902, and pNTF3-1902 POU3F2 mut. Approximately 24 h later, cells were treated with neuronal induction medium. The transcriptional activity of each reporter was normalized to the Renilla luciferase activity and compared with that of cells transfected with the pGL3 basic vector (to which a value of 1 was assigned). *** p < 0.001. Values are presented as mean ± SEM of three independent experiments for b and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

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

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of POU3F2 silencing on neuronal differentiation and NTF3 expression in NT2D1 cells. a POU3F2 expression in POU3F2-knockdown (shPOU3F2) and control (shLuc) NT2D1 cells was determined by Western blot analyses after neuronal induction for 6 h. GAPDH was used as a loading control. The values represent the relative expression compared with that of the non-induced shLuc cells (to which a value of 1 was assigned). b NTF3 mRNA expression of the cells described in a was analyzed by real-time PCR. mRNA levels were calculated relative to that of the non-induced shLuc cells. * p < 0.05; *** p < 0.001. c Neuronal morphology of shLuc and shPOU3F2 cells that were treated with neuronal induction medium for 24 h or left untreated (non). d Quantification of cell numbers of shLuc and shPOU3F2 described in c . All the percentages of the shLuc and shNTF3 cells were compared to that of the non-induction shLuc cells (to which a value of 100% was assigned). e β3-tubulin staining was performed on shLuc and shPOU3F2 cells, which were treated with neuronal induction medium for 0, 6, or 24 h or left untreated, after which neuronal cells were detected. Values represent the mean ± SEM of three independent experiments for b and d

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

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

Journal: Molecular Neurobiology

Article Title: NTF3 Is a Novel Target Gene of the Transcription Factor POU3F2 and Is Required for Neuronal Differentiation

doi: 10.1007/s12035-018-0995-y

Figure Lengend Snippet: Effects of NTF3 silencing and NTF3 recombinant protein treatment on the viability and neuronal differentiation of NT2D1 cells. a NTF3 mRNA levels in NTF3-knockdown (shNTF3) and control (shLuc) NT2D1 cells, which were treated with neuronal induction medium for 0, 24, or 48 h or left untreated (Non), were determined by real-time PCR. mRNA levels were calculated as the relative expression compared with the untreated shLuc cells. *** p < 0.001. b Phase contrast microscopy images of untreated shLuc and shNTF3 cells and those cells 24 h after neuronal induction with concomitant treatment of rNTF3 (5, 20 ng/ml) or vehicle. c Quantification of neuron number of shLuc and shNTF3 cells as described in b . All the percentages of neurons differentiated from shLuc and shNTF3 cells were compared to that of neurons differentiated from the vehicle-treated shLuc cells (to which a value of 100% was assigned). * p < 0.05; ** p < 0.01. d A suggested model of the POU3F2/NTF3 pathway that mediates the process of neuron differentiation. Values are presented as mean ± SEM of at least three independent experiments for a and c

Article Snippet: Human pluripotent embryonic carcinoma NTERA2 cl.D1 (NT2D1) cells (ATCC, CRL1973) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Recombinant, Knockdown, Control, Real-time Polymerase Chain Reaction, Expressing, Microscopy

Journal: Molecular and Cellular Biology

Article Title: p150 Sal2 Is a p53-Independent Regulator of p21 WAF1/CIP

doi: 10.1128/mcb.24.9.3885-3893.2004

Figure Lengend Snippet: FIG. 5. p150Sal2 directly interacts with p21 promoter and regulates p21 transcription through a cis-acting element. (A) Schematic representation of the 2.7-kb human p21 promoter region and its digestion fragments used in the protein DNA immunoprecipitation assay (25) (sizes in base pairs are given under each fragment). (B) Immunoprecipitation of AvaI-digested, StyI-digested, or StyI/MspI double-digested fragments of p21 promoter by p150 antibody (AntiP150) with preimmune serum (Pre-Imm) as a control. Wild-type (Wt) p150 or p150 with a deletion of the putative DNA binding domain (3) was from either P19 cell extract (Cell) or the in vitro translation (IVT) product of cloned human cDNA. Empty cloning vector was used as a negative control for in vitro-translated p150 in the immunoprecipitation of StyI/MspI fragments. (C) A p150-responsive cis-acting element is located 1.4 kb upstream of the human p21 promoter corresponding to the distal p150Sal2 binding region. A luciferase assay was conducted in SKOV-3 cells to assess the responsiveness of various p21 promoter deletion constructs. Fold induction is the corresponding luciferase activity with cotransfected p150Sal2 over that with cotransfected empty vector. The p53 binding sites are shown as landmarks. (D) p150 binds p21 promoter region in vivo. ChIP was performed using serum against p150 (Anti-p150) to precipitate chromatin cross-linked with p150 in P19 cells and amplified using p21 promoter-specific primers (p21) and primers for GAPD. The same sets of primers were also used for PCR amplification from preimmune serum-precipitated chromatin (PreImm) and total input chromatin extract (Total) as controls.

Article Snippet: SKOV-3 and P19 cells were obtained from the American Type Culture Collection.

Techniques: Immunoprecipitation, Control, Binding Assay, In Vitro, Clone Assay, Cloning, Plasmid Preparation, Negative Control, Luciferase, Construct, Activity Assay, In Vivo

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 1. Cationic AuNP combined with Ad5wtGFP enhanced viral uptake in PCa and PDAC cell lines. (A) PCa cells were infected with Ad5wtGFP at 10, 100 and 500 ppc (PC3) and 10 and 50 ppc (22Rv) in the presence or absence of 0.5 pmol AuNPs. (B) PDAC cells were infected with Ad5wtGFP at 100 ppc and the stellate PS1 cells at 500 ppc in the presence or absence of 0.2 pmol AuNPs; samples were analysed 24 and 48 h after infection. (A,B) The EGFP signal was measured as an indication of infection levels by flow cytometry, averages ±SD, n = 3, ** p < 0.01, *** p < 0.001. (C) Fluorescent images of Panc04.03, PT45 and PS1 cells 24 and 48 h after infection with Ad5wtGFP at 50 ppc with and without AuNP at 0.2 pmol, representative of 3 studies. Images were taken at 10× magnification (Olympus IX70 microscope, Evident Europe GmbH, Stansted, UK).

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: Infection, Cytometry, Microscopy

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 2. Ad∆∆-induced cell killing is enhanced by AuNPs in PCa and PDAC cells. (A) Dose–response to Ad∆∆in PC3 and 22Rv cells with and without 0.1 pmol AuNPs. Cell viability determined by MTS assay. (B) EC50-values (left panel) and the relative decreases in EC50-values (right panel) in each cell line compared to Ad∆∆alone. (C) PC3 cells treated with fixed doses of Ad∆∆at 500 ppc or 750 ppc and/or mitoxantrone (450 nM) with and without AuNPs at 0.1 pmol. Cell viability was measured by MTS assay 4d after infection, ** p < 0.01 (Ad/AuNP vs. Ad) and $$ p < 0.01 (Ad/M vs. Ad). (D) EC50-values generated from Ad∆∆dose–response curves ± AuNPs at 0.2 pmol (left panel) and the corresponding relative decreases in Panc04.03, PT45 and PS1 cells compared to Ad∆∆alone. (A–D) Averages ± SD, n = 3, * p < 0.05, ** p < 0.01.

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: MTS Assay, Infection, Generated

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 3. Ad∆∆replication is enhanced in the presence of AuNP and efficiently eliminates PDAC cells in three-dimensional co-cultures with PS1 stellate cells. (A) PC3 and 22 Rv cells were infected with Ad∆∆at 100 ppc in the presence or absence of 0.1 pmol AuNPs. PC3 cells were also treated with mitoxantrone (450 nM) and infected with Ad∆∆with and without preincubation with 0.5 pmol AuNP. (B) Panc04.03 and PT45 cells were infected at 100 pc and PS1 cells at 500 ppc with and without AuNPs at 0.2 pmol and analysed 48 h after infection. (A,B) Viral replication was determined by TCID50 assays 48 and 72 h (Pca cells) and 48 h (PDAC cells) after infection, averages ± SD, n = 3, * p < 0.05, ** p < 0.01. (C) Co-cultures of Panc04.03:PS1 (1:2) cells infected with Ad5wt (1000 or 2000 ppc) in the presence or absence of AuNP (0.5 pmol), H/E staining. Cells were cultured for 3d prior to infection with virus ± AuNPs, fixed and processed for IHC 5d post-infection, 8d-old cultures, 10× magnification, representative of three biological repeats. (D) Confocal images of co- cultures detailed in (C). Localisation of virus detected by GFP-labelled secondary antibody to the E1A-antibody (green) and nuclear DAPI stain (blue) (10× magnification).

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: Infection, Staining, Cell Culture, Virus

Journal: International journal of molecular sciences

Article Title: Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models.

doi: 10.3390/ijms23168884

Figure Lengend Snippet: Figure 4. Ad-3∆-A20T infection and replication is enhanced in the presence of AuNP in PDAC cells. (A) Panc04.03 and PT45 were infected with Ad-3∆-A20T-GFP (100 ppc) and PS1 (500 ppc) in the presence or absence of 0.2 pmol AuNPs; samples were analysed 24 and 48 h after infection by flow cytometry for GFP expression (left panel), averages ± SD, n = 3, ** p < 0.01, *** p < 0.001. Lower panel: representative fluorescent images of PS1 cells 24 h and 48 h after infection as in (A), images were taken at 10× magnification (Olympus IX70 microscope). (B) Replication rate in Panc04.03, PT45 and PS1 cells, determined by qPCR for viral genome copies. Cells were infected as above with Ad∆∆and Ad-3∆-A20T in the presence or absence of AuNPs at 0.2 pmol and viral DNA quantified after 24, 48 and 72 h. Data presented from one experiment in triplicates relative to the respective virus alone at 24 h, averages ± SEM.

Article Snippet: Human prostate cancer PC3 and 22Rv1 (ATCC, LGC Standards, UK), and human pancreatic ductal adenocarcinoma (PDAC) Panc04.03 (ATCC) and PT45 (Prof H. Kalthoff, Comprehensive Cancer Centre, Campus Kiel, Kiel, Germany) cell lines were used in the study.

Techniques: Infection, Cytometry, Expressing, Microscopy, Virus

Journal: Cell death & disease

Article Title: Reduction of spermine synthase enhances autophagy to suppress Tau accumulation.

doi: 10.1038/s41419-024-06720-8

Figure Lengend Snippet: Fig. 4 SMS knockdown upregulates autophagy and suppresses Tau accumulation in human neuronal or glial cell lines. A Western blot of Tau, EGFP, autophagy marker LC3, cargo recruiter p62 and SMS in SH-SY5Y cells with Tau/EGFP plasmids and Control/SMS siRNA transfection. The image is a representative of four separate experiments. B Quantification of the protein levels of Tau (5A6), EGFP, LC3-I (cytoplasmic), LC3-II (autophagosome-associated), p62, and SMS in (A). All the protein levels were normalized with the β-Actin level. All the values were further normalized by that of the control cells. n = 4; Student’s t test (Tau or EGFP) or two-way ANOVA Sidak’s multiple comparisons (others). C p62 staining and Alexa 647-conjugated Tau K18 fibrils in SVG p12 cells with Control/SMS siRNA transfection. The images are representatives of five fields. D, E Quantification of the size (area) (D) and intensity (E) of the Alexa 647-conjugated Tau K18 fibrils in (C). n = 5; Student’s t test. Data represent mean ± SEM.

Article Snippet: Control or SMS siRNA are transfected into SVG p12 cells (CRL-8621, ATCC) on coverslips in 12-well plates as mentioned above.

Techniques: Knockdown, Western Blot, Marker, Control, Transfection, Staining