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Inhibition of DNA repair by knockdown of PHAX. ( A ) U2OS cells were transfected with the indicated siRNAs, followed by UV-irradiation (20 J/m 2 ). After 6-h incubation, phase-contrast images were obtained. ( B , C ) U2OS cells were transfected with the indicated siRNAs, followed by ADR ( B ) or CPT ( C ) at the indicated concentrations. After 24-h incubation, cell viabilities were determined by alamarBlue assay. ( D ) U2OS cells were treated as in A . The indicated protein levels were determined by western blotting analysis. ( E – H ) U2OS cells were cotransfected with the indicated siRNAs and each reporter plasmid for DNA repair assay (pDRGFP for HDR [ E , F ] or <t>pEJ2GFP</t> for NHEJ [ G , H ]). Fluorescence microscopic images were obtained, and the numbers of GFP-positive cells were counted. As a positive control, the cells were treated with PARP inhibitor (PARPi) at 100 nM for 48 h. Data are the means ± S.D. ( n = 4). (*) P < 0.01, (**) P < 0.001.
Pej2gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Inhibition of DNA repair by knockdown of PHAX. ( A ) U2OS cells were transfected with the indicated siRNAs, followed by UV-irradiation (20 J/m 2 ). After 6-h incubation, phase-contrast images were obtained. ( B , C ) U2OS cells were transfected with the indicated siRNAs, followed by ADR ( B ) or CPT ( C ) at the indicated concentrations. After 24-h incubation, cell viabilities were determined by alamarBlue assay. ( D ) U2OS cells were treated as in A . The indicated protein levels were determined by western blotting analysis. ( E – H ) U2OS cells were cotransfected with the indicated siRNAs and each reporter plasmid for DNA repair assay (pDRGFP for HDR [ E , F ] or pEJ2GFP for NHEJ [ G , H ]). Fluorescence microscopic images were obtained, and the numbers of GFP-positive cells were counted. As a positive control, the cells were treated with PARP inhibitor (PARPi) at 100 nM for 48 h. Data are the means ± S.D. ( n = 4). (*) P < 0.01, (**) P < 0.001.

Journal: RNA

Article Title: The RNA transport factor PHAX is required for proper histone H2AX expression and DNA damage response

doi: 10.1261/rna.074625.120

Figure Lengend Snippet: Inhibition of DNA repair by knockdown of PHAX. ( A ) U2OS cells were transfected with the indicated siRNAs, followed by UV-irradiation (20 J/m 2 ). After 6-h incubation, phase-contrast images were obtained. ( B , C ) U2OS cells were transfected with the indicated siRNAs, followed by ADR ( B ) or CPT ( C ) at the indicated concentrations. After 24-h incubation, cell viabilities were determined by alamarBlue assay. ( D ) U2OS cells were treated as in A . The indicated protein levels were determined by western blotting analysis. ( E – H ) U2OS cells were cotransfected with the indicated siRNAs and each reporter plasmid for DNA repair assay (pDRGFP for HDR [ E , F ] or pEJ2GFP for NHEJ [ G , H ]). Fluorescence microscopic images were obtained, and the numbers of GFP-positive cells were counted. As a positive control, the cells were treated with PARP inhibitor (PARPi) at 100 nM for 48 h. Data are the means ± S.D. ( n = 4). (*) P < 0.01, (**) P < 0.001.

Article Snippet: pDRGFP (plasmid 26475), pEJ2GFP (44025), pCBASceI (26477) were obtained from Addgene.

Techniques: Inhibition, Knockdown, Transfection, Irradiation, Incubation, Alamar Blue Assay, Western Blot, Plasmid Preparation, Fluorescence, Positive Control