53bp1 mcherry fragment  (Addgene inc)

Journal: eLife

Article Title: DNA damage induces nuclear actin filament assembly by Formin-2 and Spire-1/2 that promotes efficient DNA repair

doi: 10.7554/eLife.07735

Figure Lengend Snippet: ( A ) Scatter plot comparing the number of 53BP1-mTagBFP2 foci per cell and 53BP1-mTagBFP2 expression level following treatment with 0.01% MMS for 120′. N = 354 cells. ( B ) Localization of Utr230-EN after 0.01% MMS treatment following transient siRNA knockdown of exportin-6 (XPO6) or IPO9. ( C ) Quantification of elongated nuclear filament formation after increasing doses of MMS in IPO9, XPO6, and control siRNA knockdown lines. N = 126–150 cells. ( D ) Quantification of DSBs per cell detected by 53BP1 antibody after increasing doses of MMS in IPO9, XPO6, and control knockdown cell lines. N = 126–150 cells. Asterisks indicate p-values < 10E-2 (*) or 10E-3 (**). DOI: http://dx.doi.org/10.7554/eLife.07735.007

Article Snippet: 53BP1 fragment was subcloned from Addgene plasmid #19835 ( ) and was provided by Beth Cimini. mCherry-FMN2 was provided by Sonia Rocha and includes the following N-terminal insertion in comparison to the NCBI human FMN2 reference sequence (accession number NP_064450.3): AGATCTCATTCGATTCGCACGGTGGAGATTAAAGTCCCCGAGATAGAGGAAACGTTTTTCGCGCCCAGGTTCAGCGAGGAGCCGCGCGGGGGCAGAGGGGGCGGCGGCGGCGGGCGGGGAGCCAGGCCCGAGCTGCGTTCTGCGCAGCCATTGGTGGGCGCCGCACTCTGCACTGAGCATGTTCGCGCCCCGCCGGCCCCTAGCCGCAGCCGCAGCCGCAGCGACGGCAGCCACGGGAGCCGCCGCGCATTATGCAAAGCGGCGGCAGATGCGAGCGGGGCCAGCCGGGCGCGCGTCGGCCTCCCCTCCCAGCGGCTCCCCCCGCCGCCGCCTGACTCTCCCGGGAGACTCCCTAGGCCCGGGATTGCACC. roGFP2 was a gift from Philip Merksamer and Ferroz Papa.

Techniques: Expressing, Knockdown, Control

Journal: eLife

Article Title: DNA damage induces nuclear actin filament assembly by Formin-2 and Spire-1/2 that promotes efficient DNA repair

doi: 10.7554/eLife.07735

Figure Lengend Snippet: ( A ) Double-strand break (DSB) sites detected by 53BP1 immunofluorescence after 30′, 60′, 90′, and 120′ incubations in 0.01% MMS. ( B ) Average number of DSB foci detected by 53BP1 immunofluorescence per cell after stable shRNA knockdown of the nuclear actin import factors, importin-9 (IPO9) or cofilin, after 0.01% MMS incubation. N = 125–172 cells per condition. ( C ) Average number of DSB foci detected by gamma H2AX immunofluorescence per cell after stable shRNA IPO9 knockdown following 0.01% MMS incubation. N = 182–241 cells per condition. ( D ) Partial rescue of control DSB foci levels after IPO9 knockdown by overexpression of wild-type actin-NLS-P2A-mCherry but not non-polymerizing R62D mutant of actin-NLS-P2A-mCherry. N = 118–150 cells per condition. ( E ) Full rescue of non-Target control DSB foci levels after IPO9 knockdown by overexpression of wild-type actin-NLS-P2A-mCherry but not non-polymerizing R62D mutant of actin-NLS-P2A-mCherry. N = 262–291 cells per condition. ( F ) Comparison of the distributions of long (>1 micron) nuclear filaments per cell and 53BP1 foci counts. N = 206 cells. ( G ) Co-localization assays between Utr230-EN and DSBs after 120′ incubation in 0.01% MMS. Asterisks indicate p-values < 10E-2 (*), 10E-3 (**), or 10E-4 (***) for all panels. DOI: http://dx.doi.org/10.7554/eLife.07735.006

Article Snippet: 53BP1 fragment was subcloned from Addgene plasmid #19835 ( ) and was provided by Beth Cimini. mCherry-FMN2 was provided by Sonia Rocha and includes the following N-terminal insertion in comparison to the NCBI human FMN2 reference sequence (accession number NP_064450.3): AGATCTCATTCGATTCGCACGGTGGAGATTAAAGTCCCCGAGATAGAGGAAACGTTTTTCGCGCCCAGGTTCAGCGAGGAGCCGCGCGGGGGCAGAGGGGGCGGCGGCGGCGGGCGGGGAGCCAGGCCCGAGCTGCGTTCTGCGCAGCCATTGGTGGGCGCCGCACTCTGCACTGAGCATGTTCGCGCCCCGCCGGCCCCTAGCCGCAGCCGCAGCCGCAGCGACGGCAGCCACGGGAGCCGCCGCGCATTATGCAAAGCGGCGGCAGATGCGAGCGGGGCCAGCCGGGCGCGCGTCGGCCTCCCCTCCCAGCGGCTCCCCCCGCCGCCGCCTGACTCTCCCGGGAGACTCCCTAGGCCCGGGATTGCACC. roGFP2 was a gift from Philip Merksamer and Ferroz Papa.

Techniques: Immunofluorescence, shRNA, Knockdown, Incubation, Control, Over Expression, Mutagenesis, Comparison