Journal: Longevity & Healthspan

Article Title: The secreted protein S100A7 (psoriasin) is induced by telomere dysfunction in human keratinocytes independently of a DNA damage response and cell cycle regulators

doi: 10.1186/2046-2395-3-8

Figure Lengend Snippet: Colony-forming efficiency analysis of NHEK transduced with p14 ARF , p16 INK4A , p53 and TRF2 ΔBΔM . NHEKs were transduced with amphotropic retroviral particles using spinfection and, 48 h later, trypsinised and seeded at clonal density (7 × 10 3 cells per 6-well plate). Cells were cultured for 2 weeks under drug selection and finally fixed and stained with Rhodamine B to reveal keratinocyte colonies. Colony-forming efficiency, displayed as percentage and relative to the respective EV control, was calculated by dividing the total number of colonies obtained per well by the total number of cells seeded per plate (7,000). Photos show wells representative of the results obtained for each construct. Legend: GFP, empty vector control for p14 ARF , p16 INK4A and p53 ; EV, empty vector control for TRF2 ΔBΔM (DN). This is the result of a single experiment.

Article Snippet: Retroviral vectors pLPC-N MYC (12540 Addgene, Cambridge, MA.) and pLPC-NMYC TRF2 ΔBΔM (16069 Addgene) were donated by Titia de Lange (Rockefeller University, NYC, USA); pBABE-puro p14 ARF , pBABE-puro p16 INK4a and pBABE-puro p53 were donated by Gordon Peters (London Research Institute, CRUK, London, UK); and pBABE-puro GFP was donated by Cleo Bishop (Blizard Institute, QMUL, London, UK).

Techniques: Transduction, Retroviral, Cell Culture, Selection, Staining, Control, Construct, Plasmid Preparation

Journal: Cancer Science

Article Title: MicroRNA‐493‐5p‐mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

doi: 10.1111/cas.14292

Figure Lengend Snippet: Measurement of microRNA (miR)‐493‐5p and MYCN expression levels in clinical samples from hepatocellular carcinoma (HCC) patients. A,B, Relative expression levels of (A) miR‐493‐5p and (B) MYCN in clinical samples. Patients showing moderate to advanced hepatic tumors (length > 2.5 cm) were selected for the study. The box plots illustrate differential gene expression in 13 primary HCC tumors (T) compared with the corresponding nontumor tissues (NT). Mann–Whitney U test was used to calculate P values. C, Scatter plots of Spearman’s correlation coefficient analysis between miR‐493‐5p and MYCN relative expression, measured by real‐time quantitative PCR in all clinical samples (T and NT, N = 26). Red and blue plots show T and NT tissues, respectively

Article Snippet: For MYCN rescue experiments, the cells were incubated with 1.5 µg MYCN expression vector (plasmid #74163; Addgene) following the experimental procedure described above.

Techniques: Expressing, MANN-WHITNEY, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: MicroRNA‐493‐5p‐mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion

doi: 10.1111/cas.14292

Figure Lengend Snippet: Effect of MYCN knockdown and rescue on hepatic cancer cell growth and invasion. A, Hep3B cell growth assessment after MYCN silencing. Two distinct siRNAs were used to target MYCN (siMYCN_A and siMYCN_B). Scrambled siRNA was used as a negative control (siCtrl). Number of cells was estimated at the indicated times using a cell viability assay. B, Invasive abilities of Hep3B cells after MYCN knockdown. Cells that migrated through the Matrigel‐coated membrane were counted after 72 h. Photographs are representative of cell invasion for each condition. C, Cell viability. D, Invasion assay after MYCN and microRNA (miR)‐493‐5p overexpression in Hep3B cells (rescue experiment). MYCN expression vector did not contain MYCN mRNA 3′‐UTR. Cell viability was measured after 4 d. Validation of MYCN expression vector compared with the mock is shown in Figure . Data depicted in the figure show the mean ± SD. Significant differences were evaluated with a t test (n = 3). * P < .05, ** P < .01, *** P < .001

Article Snippet: For MYCN rescue experiments, the cells were incubated with 1.5 µg MYCN expression vector (plasmid #74163; Addgene) following the experimental procedure described above.

Techniques: Negative Control, Viability Assay, Invasion Assay, Over Expression, Expressing, Plasmid Preparation

Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: ( A ) Western blotting analyses of the indicated pro-homologous recombination (HR) factors in total cell extracts from GCT27 paired cell lines. Graph bars quantify protein level differences among GCT27 cell lines. Data are mean values ± s.d. of either three (breast cancer type 1 susceptibility protein, BRCA1) or six (C-terminal binding protein-1 interacting protein, CtIP) independent experiments. Original blots see . Statistical analyses were performed using a two-tailed t -test (* p < 0.05; ** p < 0.01). ( B ) Western blotting analyses of the indicated pro-HR factors in chromatin extracts from GCT27 paired cell lines. Cells were treated with a pulse of 3 μM cisplatin for 6 h and analyzed 16 h after culture in drug-free media. Histone H3 (H3) was used as a loading control. Original blots see . ( C ) Quantification of BRCA1 foci number in GCT27 paired cell lines before (Ctr) and after treatment with 3 μM cisplatin. Treated cells were collected both at the end of treatment (6 h) and 16 h after drug washout. Data are mean values ± s.d. of two independent experiments, each performed in duplicate. ( D ) Western blotting analyses of the indicated pro-HR factors in total cell extracts of 2102EP paired cell lines. Tubulin and cyclin A were assessed to detect differences in protein loading and cell cycle phase distribution, respectively. Original blots see . ( E ) Western blotting analyses of the indicated pro-HR factors in chromatin extracts from the indicated cell lines, before and after treatment with 3 μM cisplatin for 6 h. Cell extracts were prepared from cells collected 16 h after drug removal. H3 was used as a loading control. Original blots see . ( F , G ) Western blotting analyses of the indicated pro-non-homologous end joining (NHEJ) factors in total cell extracts of 2102EP and GCT27 cell lines. Clathrin was used as a loading control for 53BP1 and DNA-PKcs, while KU70 expression was normalized using tubulin. Graph bars quantify protein levels difference among cell lines. Original blots see . Data are mean values ± s.d. of six (53BP1) or three (DNA-PKcs) independent experiments. In all quantifications, the expression of the indicated proteins was normalized on the expression of either tubulin or clathrin. Cyclin A was assessed to detect possible differences in cell cycle phase distribution among cell lines. Statistical analyses were performed using a one-tailed t -test (* p < 0.05; ** p < 0.01; **** p < 0.0001). A.U. = arbitrary units.

Article Snippet: To stably increase the expression of 53BP1 in 2102EPcis-r cells, we infected them using retroviral particles expressing either the N-Myc-53BP1 WT pPLC-Puro vector (Addgene, Watertown, NY, USA, 19836) or pLPC-N MYC (Addgene, 12540) as a control.

Techniques: Western Blot, Homologous Recombination, Binding Assay, Two Tailed Test, Non-Homologous End Joining, Expressing, One-tailed Test

Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: ( A , B ) NHEJ repair proficiency of GCT27 and 2102EP cell lines as measured by the EJ5-GFP reporter substrate. NZE-GFP indicates the transfection efficiency of each cell line. Data are mean values ± s.d. of three independent experiments. ( C , D ) Colony assay of the indicated cell lines treated with the indicated doses of X-rays. The surviving fraction was monitored by following colony formation for up to 10 to 14 days after treatment. Data are mean values ± s.d. of two independent experiments, each performed in triplicate. ( E , F ) Quantification of 53BP1 foci in S/G2 (cyclin A-positive) nuclei of the indicated cell lines after X-ray treatment. Data are mean values ± s.d. of three independent experiments. ( G , H ) Quantification of 53BP1 foci in S/G2 nuclei of the indicated cell lines after cisplatin treatment. Cells were treated with a 6 h pulse of 3 μM cisplatin and collected at the end of stimulation and 16 h after drug washout. Data are mean values ± s.d. of three independent experiments. Statistical analyses were performed using an unpaired two-tailed ( A , B , E – H ) or one-tailed ( C , D ) t -test (* p < 0.05; ** p < 0.01).

Article Snippet: To stably increase the expression of 53BP1 in 2102EPcis-r cells, we infected them using retroviral particles expressing either the N-Myc-53BP1 WT pPLC-Puro vector (Addgene, Watertown, NY, USA, 19836) or pLPC-N MYC (Addgene, 12540) as a control.

Techniques: Transfection, Colony Assay, Two Tailed Test, One-tailed Test

Journal: Cancers

Article Title: Testicular Germ Cell Tumors Acquire Cisplatin Resistance by Rebalancing the Usage of DNA Repair Pathways

doi: 10.3390/cancers13040787

Figure Lengend Snippet: ( A ) Western blotting analyses of 53BP1 expression in 2102EP cells. Here, #1, #2, #3 are clones of 2102EPcis-r cells overexpressing 53BP1 at a level comparable to that of cis-s cells. Ctr indicates the level of 53BP1 protein in naïve 2102EPcis-r cells. Tubulin was used as loading control. Original blots see . ( B ) Colony assay of control and 53BP1-overexpressing clones of 2102EPcis-r after treatment with a pulse of 3 μM cisplatin for 6 h. Ctr = cells transfected with a control vector; Unt. = untreated; cisp. = cisplatin-treated. ( C ) Quantification of surviving colonies of the indicated 2102EPcis-r cells, treated as described in ( B ). ( D ) Western blotting analyses of 53BP1 expression in GCT27 cells. GCT27cis-s and GCT27cis-r are non-infected paired clones. GCT27cis-s ShRNACtr are cells infected with a non-specific shRNA, while GCT27cis-s ShRNAmir1 are cells infected with an shRNA specific to 53BP1. Tubulin was used as a loading control. Original blots see . ( E ) Colony assay of GCT27 cell lines infected with either ShRNACtr or ShRNAmir1. Data are mean values ± s.d. of three independent experiments. ( F , G ) Surviving fraction of the indicated cell lines after treatment with either cisplatin or cisplatin combined with DNA-PKi. Survival was evaluated by crystal violet assay. Data are mean values ± s.d. of either three (GCT27) or six (2102EP) independent experiments. Statistical analyses were performed using the unpaired two-tailed ( C , F , G ) or one-tailed ( E ) t -test (* p < 0.05; ** p < 0.01; *** p < 0.0001).

Article Snippet: To stably increase the expression of 53BP1 in 2102EPcis-r cells, we infected them using retroviral particles expressing either the N-Myc-53BP1 WT pPLC-Puro vector (Addgene, Watertown, NY, USA, 19836) or pLPC-N MYC (Addgene, 12540) as a control.

Techniques: Western Blot, Expressing, Clone Assay, Colony Assay, Transfection, Plasmid Preparation, Infection, shRNA, Crystal Violet Assay, Two Tailed Test, One-tailed Test

Journal: Oncotarget

Article Title: Protein arginine methyltransferase 1 is a novel regulator of MYCN in neuroblastoma

doi: 10.18632/oncotarget.11556

Figure Lengend Snippet: ( A ) Prmt1 fl/− ER-Cre MEF cells were transfected with pcDNA3.1 V5-MYCN. Two days after transfection, the whole cell lysates were subjected to immunoprecipitation with ASYM24 antibody, followed by western blot with anti-V5 antibody. ( B ) (left) Prmt1 fl/− ER-Cre MEF cells were induced with OHT for 4 days, followed by transfection with pcDNA3.1 V5-MYCN for 2 days, and analyzed for IP with ASYM24 antibody. (right) Western blot analysis of whole cell lysates with anti-V5 and anti-PRMT1 (in a separate repeat blot) antibodies. β-actin was used as a loading control. ( C ) In vitro methylation assays with recombinant PRMT1 together with GST or GST-MYCN full-length proteins in the presence of [ 3 H] SAM. A non-specific band close to 37 kDa is indicated, most likely derived from co-purified proteins with recombinant PRMT1 from insect cells. ( D ) In vitro methylation assays with recombinant PRMT1 together with GST or GST-MYCN (51–70) fusion proteins (WT or R65A) in the presence of [ 3 H] SAM.

Article Snippet: The human MYCN cDNA expression construct was subcloned from pTH-MYCN, a gift from William A. Weiss (Addgene plasmid # 35416) [ ], into pcDNA 3.1 vector to make pcDNA3.1-V5-MYCN, and into pGEX-5X-1 to make pGEX-5X-V5-MYCN.

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Methylation, Recombinant, Derivative Assay, Purification

Journal: Bioengineered

Article Title: Retracted article: MYCN contributes to the sensitization of acute myelogenous leukemia cells to cisplatin by targeting SRY-box transcription factor 4.

doi: 10.1080/21655979.2021.1997697

Figure Lengend Snippet: Figure 7. Knockdown of MYCN retarded the AML tumor growth in mice. The BALB/c nude mice were injected with vector- or shMYCN-transfected HL60 cells. (a) The images of formed tumors were captured. (b) The tumor size was measured and calculated every three days. (c) At 27 days postinjection, the mice were euthanized, and the tumor was resected and weighted. (d) The IHC assay was administrated to quantify the MYCN and SOX4 expressions in tumors. Scale bar: 100 μm. ^^^P < 0.001, vs. Vector. (MYCN, MYCN proto-oncogene; AML, acute myeloid leukemia; shMYCN, small hairpin RNA for MYCN; SOX4, SRY-Box Transcription Factor 4, IHC, immunohistochemistry).

Article Snippet: The overexpression plasmid of MYCN (RC201241, Origene), empty pCMV6 vector (PS100001, Origene), small interfering RNA (siRNA) for MYCN (siMYCN, SR321048), SOX4 (siSOX4, SR304530) and their negative controls (siNC, SR30002), shMYCN (TL316627), as well as empty pGFP-C-shLenti shRNA vector (TR30023) were purchased from Origene (Rockville, Maryland, USA).

Techniques: Knockdown, Injection, Plasmid Preparation, Transfection, Immunohistochemistry