Fig. 1: Rapid detection of cellular HR and SSA activity by transient DNA transfection.
From: HR eye & MMR eye: one-day assessment of DNA repair-defective tumors eligible for targeted therapy
a Schematic of Nluc-based DNA constructs to detect HR (HR-Nluc-v1) and SSA (SSA-Nluc-v1). Horizontal arrow, CMV promoter; gray oval, polyA signal. b Monitoring of luciferase activity in human HT1080 and Nalm-6 cell lines following transfection. c Comparison of HR and SSA activity in 6 different human cell lines. Luciferase activity was measured 24 h after transfection, and relative luciferase activity (pCMV-Nluc was taken as 1) was calculated from the data shown in b and Supplementary Fig. 3c. Data shown are the mean ± s.d. of three independent experiments, except for SSA analysis using HeLa S3 and U2OS. d Impact of Rad51 knockdown on extrachromosomal HR and SSA in HT1080 and Nalm-6 cells. Data shown are the mean ± s.d. of three independent experiments. e Impact of BRCA1/BRCA2 deficiency on extrachromosomal HR and SSA. Data shown are the mean ± s.d. of three independent experiments. f Schematic of HR-Nluc-v2. Note that imperfect Nluc fragments are placed on separate DNA molecules, with iNluc-carrying DNA in a circular form so that HR dominates over SSA (see Supplementary Fig. 5f). g Impact of HR deficiency on extrachromosomal HR of HR-Nluc-v1 and HR-Nluc-v2. The relative luciferase activity (HR-deficient vs HR-proficient) is shown in (d and e). Symbols are as in (d and e). Data shown are the mean ± s.d. of three independent experiments. h Analysis of HR status of MDA-MB-436-derived olaparib-resistant clones. Data shown are the mean ± s.d. of four independent experiments. Note that shown are the results of 4 out of 11 olaparib-resistant clones, and the results of all the resistant clones are shown in Supplementary Fig. 7b. i Analysis of HR status of Myc/Mycn-overexpressing cells harboring MYC/MYCN gene amplification. Data shown are the mean ± s.d. of three independent experiments. Statistical significance was determined by two-sided Student’s t-test (d, e, and g), or by one-way ANOVA with Dunnett’s multiple comparisons (h and i). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Source data are provided as a Source Data file.
