plasmid 50 ng  (New England Biolabs)

Journal: Science Advances

Article Title: RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer

doi: 10.1126/sciadv.abl9794

Figure Lengend Snippet: ( A ) Comparison of E2F1 transcript levels between RNASEH2B/RB1 co–wild-type (co-WT) and co-deletion (co-DEL) tumors in three PCa clinical cohorts in cBioPortal. ( B ) Comparison of E2F1 transcriptional activity between SKO and DKO cells in the presence or absence of olaparib as indicated for 24 hours using E2F1 luciferase reporter assay. ( C ) Publicly available E2F1 ChIP-seq data showing E2F1 binding capacity at the promoters of BRCA1/2 and RAD51 genes in LNCaP cells. The E2F1 ChIP-seq peaks were observed in the UCSC Genome Browser. Red arrows indicate the qPCR regions. ( D ) E2F1 binding was determined by ChIP-qPCR at the promoters of BRCA1/2 and RAD51 genes in C4-2B and 22Rv1 cells. An irrelevant genomic region was used as a control. Normal IgG and anti-E2F1 antibody were used for immunoprecipitation. ( E ) Western blots show protein levels of indicated genes in DKO C4-2B and 22Rv1 cells after E2F1 siRNA knockdown or the treatment with pan-E2F inhibitor HLM006474 for 24 hours. β-Actin serves as a loading control. The intensity of protein bands was quantified using ImageJ software. The first band was defined as 1. ( F ) BRCA1/2 and RAD51 mRNA levels were determined by RT-qPCR in DKO C4-2B and 22Rv1 cells in comparison to control and SKO cells. ( G ) Western blots show protein levels of BRCA1/2 and RAD51 in SKO and DKO cells after olaparib treatment for 24 hours. Western blot quantification is described in (E). ( H ) Cell cycle distribution was analyzed in AAVS1 control, SKO, and DKO C4-2B and 22Rv1 cells under regular cell culture condition. P values were determined by two-tailed t test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Cells (1 × 10 4 cells per well) were seeded in 96-well plates and transfected with E2F1 luciferase reporter plasmids (50 ng per well) using X-tremeGENE HP DNA Transfection Reagent (#06366236001, Sigma-Aldrich).

Techniques: Activity Assay, Luciferase, Reporter Assay, ChIP-sequencing, Binding Assay, Immunoprecipitation, Western Blot, Software, Quantitative RT-PCR, Cell Culture, Two Tailed Test

Journal: Science Advances

Article Title: RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer

doi: 10.1126/sciadv.abl9794

Figure Lengend Snippet: ( A ) RNASEH2B/RB1 DKO 22Rv1 cells were injected subcutaneously into ICR-SCID mice. Mice were randomly assigned into four groups ( n = 5 animals per group) and treated with vehicle, olaparib (50 mg/kg), VE-822 (25 mg/kg), or olaparib in combination with VE-822 for three cycles as indicated. Both drugs were administered by oral gavage once a day. Tumor volume and mouse weight were recorded and analyzed across four groups as indicated. ( B ) DKO C4-2B and 22Rv1 cells were treated with DMSO, olaparib, VE-822, or olaparib + VE-822 for 24 hours. E2F1 activity was detected using E2F1 luciferase reporter assay. ( C ) Western blots show protein levels of BRCA1/2 and RAD51 in DKO cells after the treatment with DMSO, olaparib, VE-822, or olaparib + VE-822 for 24 hours. The intensity of protein bands was quantified using ImageJ software. The first band was defined as 1. ( D ) Representative images of immunofluorescence staining for RAD51 foci in DKO C4-2B and 22Rv1 cells after the treatment with DMSO, olaparib (10 μM), VE-822 (1000 nM), or olaparib + VE-822 for 24 hours. RAD51 foci were counted in at least 50 cells for each replicate under each condition ( n = 3 biological replicates). Scale bars, 20 μm. ( E ) Schematic model depicting the mechanism by which concurrent deletions of RNASEH2B , RB1 , and BRCA2 genes affect the response to PARP inhibition. P values were determined by two-tailed t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells (1 × 10 4 cells per well) were seeded in 96-well plates and transfected with E2F1 luciferase reporter plasmids (50 ng per well) using X-tremeGENE HP DNA Transfection Reagent (#06366236001, Sigma-Aldrich).

Techniques: Injection, Activity Assay, Luciferase, Reporter Assay, Western Blot, Software, Immunofluorescence, Staining, Inhibition, Two Tailed Test