Journal: Cancers

Article Title: Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting

doi: 10.3390/cancers12071939

Figure Lengend Snippet: Assessment of HRR and NHEJ in the NIH-OVCAR3 cells. ( A ) Cells were exposed to 0.5% DMSO (control) or 10 µM rucaparib for 48 h before being fixed and stained as per the protocol outlined in the methods. After imaging, the number of γH2AX and RAD51 foci per nucleus was calculated using ImageJ software, data points represent number of foci in an individual nucleus. The fold change in number of foci per nuclei between control and 10 µM rucaparib was then calculated. ( B ) Cells were co-transfected with pDRGFP and pCBASceI plasmids and analysed for the presence of GFP-positive cells 48 h post-transfection. % HRR activity was calculated by normalizing to the untransfected control. Graph shows mean of 2 individual experiments. ( C ) Cells were co-transfected with pimEJ5GFP and pCBASceI plasmids and analysed for the presence of GFP-positive cells 48 h post-transfection. % NHEJ activity was calculated by normalizing to the untransfected control. Graph shows mean of two individual experiments. [* p < 0.05; ** p < 0.005; **** p < 0.0001 (unpaired t -test)].

Article Snippet: pDRGFP (plasmid # 26475, Addgene, Watertown, MA, USA) and pCBASceI (plasmid # 26477, Addgene) plasmids used for the HRR assay were a kind gift from Maria Jasin [ , ].

Techniques: Control, Staining, Imaging, Software, Transfection, Activity Assay

Journal: Cell Death & Disease

Article Title: CHK1 and RAD51 activation after DNA damage is regulated via urokinase receptor/TLR4 signaling

doi: 10.1038/cddis.2016.291

Figure Lengend Snippet: PLAUR expression promotes DNA repair. ( a ) DNA repair assay using plasmid substrates pDRGFP (for HR) and pEJ2GFP (for NHEJ), in WT and PLAUR-expressing HEK-293 cells. Cells were transfected with pCBASceI for the introduction of a double-strand break and DNA repair efficiency correlating with GFP expression was assessed after 3 days by FACS. Data shown as mean±S.D. ( b ) MDA-MB-231 were irradiated at 5 Gy and fixed after indicated time points, kinetics of γ -H2AX foci formation was then studied by performing immunofluorescence. The number of foci per cell was quantified using ImageJ. Data shown as mean ±S.D. ( c ) Representative images of γ -H2AX foci. Scale bar 20 μm

Article Snippet: pDRGFP (Addgene, Cambridge, MA, USA, plasmid # 26475) and pCBASceI (Addgene plasmid # 26477) were a gift from Maria Jasin; EJ2GFP-puro (Addgene plasmid # 44025) was a gift from Jeremy Stark. pLenti6/V5-p53_wtp53 plasmid was a gift from Bernard Futscher (Addgene plasmid # 22945); pLKO-p53-shRNA-427 was a gift from Todd Waldman (Addgene plasmid # 25636); pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453).

Techniques: Expressing, Plasmid Preparation, Transfection, Irradiation, Immunofluorescence

Journal: Cell Death & Disease

Article Title: CHK1 and RAD51 activation after DNA damage is regulated via urokinase receptor/TLR4 signaling

doi: 10.1038/cddis.2016.291

Figure Lengend Snippet: PLAUR/TLR4 interaction increases DNA repair efficiency. ( a ) PLAUR was immunoprecipitated from HeLa cell lysates after Dox treatment, TLR4 was detected in the immunoprecipitates by western blotting. Normalization was performed by the amount of TLR4 present in the original lysates. The experiment was repeated in the reverse direction (Right). ( b ) HeLa cells were treated with radiation of 9 Gy and fixed at the indicated time points, Duolink proximity ligation assay was performed to determine the interaction between PLAUR and TLR4. Duolink signal per cell was counted and represented as a graph (right). Scale bar 20 μ m. Data shown as mean ±S.D. ( c ) DNA repair assay using plasmid substrates pDRGFP (for HR) and pEJ2GFP (for NHEJ), in WT and PLAUR-expressing HEK-Blue hTLR4cells having constitutive expression of TLR4, CD14 and MD-2. Cells were transfected with pCBASceI for the introduction of a double-strand break and DNA repair efficiency correlating with GFP expression was assessed after 3 days by FACS. Data shown as mean ±S.D.

Article Snippet: pDRGFP (Addgene, Cambridge, MA, USA, plasmid # 26475) and pCBASceI (Addgene plasmid # 26477) were a gift from Maria Jasin; EJ2GFP-puro (Addgene plasmid # 44025) was a gift from Jeremy Stark. pLenti6/V5-p53_wtp53 plasmid was a gift from Bernard Futscher (Addgene plasmid # 22945); pLKO-p53-shRNA-427 was a gift from Todd Waldman (Addgene plasmid # 25636); pLKO.1 puro was a gift from Bob Weinberg (Addgene plasmid # 8453).

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Plasmid Preparation, Expressing, Transfection