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