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: Scientific Reports

Article Title: Robust and efficient knock-in in embryonic stem cells and early-stage embryos of the common marmoset using the CRISPR-Cas9 system

doi: 10.1038/s41598-018-37990-w

Figure Lengend Snippet: Evaluation of KI efficiency for an inactive gene. ( a ) Schematic diagram of the PLP1-EGFP construct. The PLP1-EGFP TV harboured 3.1-kb and 5.0-kb homology arms, which consisted of the region surrounding the initial codon of the marmoset PLP1 gene locus. EGFP coding sequence was fused to the 3′ end of the 5′-UTR of the PLP1 gene. Polyadenylation signal sequence ( pA ) was introduced downstream of the terminal codon of EGFP . Additionally, loxP-flanked PGK-Neo-pA was placed between EGFP-pA and the 3′-homology arm. The TV was not detected by gRNAs targeting the vicinity of PLP1 exon 1 (PLP-1, 2, 3, 4, shown as scissors). Thin black arrows indicate the location of the 5′-external primer and the 3′-internal primer used for genotyping PCR of the 5′-region. Grey arrows indicate the 5′-primer homologous to the selection cassette and the 3′-external primer used for genotyping PCR of the 3′-region; b, a restriction enzyme site ( Bgl II); p, the 5′-external probe for Southern blotting. ( b ) The number of G418-resistant colonies following G418 selection of 1 × 10 6 transfected cjESCs, shown as the mean + s.e.m., n = 3. ( c ) 5′-region genotyping PCR of G418-resistant cjESC clones. Cas9+ clone numbers are shown in grey (#17–22), and Cas9− are shown in black (#17–22). Results for the remaining clones are shown in Supplementary Fig. . Ho, homozygous KI; He, heterozygous KI. ( d ) Southern blotting analysis of G418-resistant Cas9− clones using the 5′-external probe. ( e ) Summary of the PLP1 exon 1 gene targeting data. * P < 0.05, ** P < 0.01.

Article Snippet: For gene targeting experiments in the embryos, the PGK-PuroTK cassette was removed from the PLP1 -P15L vector by Cre recombinase (NEB).

Techniques: Construct, Sequencing, Selection, Southern Blot, Transfection, Clone Assay

Journal: Scientific Reports

Article Title: Robust and efficient knock-in in embryonic stem cells and early-stage embryos of the common marmoset using the CRISPR-Cas9 system

doi: 10.1038/s41598-018-37990-w

Figure Lengend Snippet: Evaluation of KI efficiency in the early-stage marmoset embryo. ( a ) Schematic of the gene targeting experiment using early-stage marmoset embryos. 2PN, the two-pronuclear stage. Scale bar, 50 μm. ( b ) Schematic of the PLP1 -P15L KI construct in the embryos. Grey arrowhead indicates the location of sequence resulting in the P15L substitution and the Sac I site in the TV. Scissors indicate the DSB site. Thin blue arrow indicates the gRNA-targeted sequence (including the PAM sequence of streptococcus pyogenes Cas9 ; NGG). Black arrowhead indicates the Cas9-cleaved sequence site. ( c ) RFLP analysis of the microinjected embryos (#12 and #14–17, the remaining results are shown in Supplementary Fig. ). White arrowheads indicate detection of the KI allele by Sac I digestion of the PCR fragments. ( d ) DNA sequence analysis of the KI allele of embryos #12 and #14. Results for the remaining embryos are appended in Supplementary Fig. .

Article Snippet: For gene targeting experiments in the embryos, the PGK-PuroTK cassette was removed from the PLP1 -P15L vector by Cre recombinase (NEB).

Techniques: Construct, Sequencing

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: eLife

Article Title: Structure of the human heparan-α-glucosaminide N -acetyltransferase (HGSNAT)

doi: 10.7554/eLife.93510

Figure Lengend Snippet:

Article Snippet: The synthesized gene was then cloned into the pEG BacMam expression vector (Addgene plasmid # 160683) between EcoRI and NotI restriction sites, to be expressed via baculoviral transduction in HEK293S GnTI - cells (ATCC # CRL-3022) as a fusion protein containing an N-terminal Strep-tag-II-GFP.

Techniques: Sequencing, Expressing, Transfection, Construct, Recombinant, Plasmid Preparation, Cloning, Modification, Software, Cell Culture, Size-exclusion Chromatography, Affinity Purification, Electron Microscopy