Journal: Nucleic Acids Research

Article Title: RNF8 has both KU-dependent and independent roles in chromosomal break repair

doi: 10.1093/nar/gkaa380

Figure Lengend Snippet: RNF8 promotes ALT-EJ. ( A ) RNA analysis of an Rnf8-/- mESC line generated using Cas9 and sgRNAs targeting Rnf8 . Shown are RT-PCR amplification products for a region of the RNF8 mRNA from exons 1–6 (Ex1–Ex6), and Actin control, for RNA from WT and Rnf8-/- mESCs, treated with and without reverse transcriptase (RT). ( B ) The Rnf8-/- mESC line shows a defect in 53BP1 foci formation. Shown are representative fluorescent images of WT and Rnf8-/- mESCs treated with 6 Gy and recovered for 4 h, stained with DAPI and antibodies for γH2AX and 53BP1. Scale bar = 20 μm. Also shown are violin plots depicting the number of γH2AX foci per nucleus (left) and 53BP1 foci per nucleus (right) in WT and Rnf8-/- mESC. N = 120. (*) P < 0.001 for WT versus Rnf8-/- measuring γH2AX or 53BP1 foci using the Mann–Whitney test. ( C ) RNF8 promotes ALT-EJ, and inhibits EJ without indels (i.e. C-NHEJ). Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, EJ6-GFP+TREX2, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, Rnf8-/- mESCs and Rnf8-/- mESCs transfected with a 3×Flag-tagged RNF8 expression vector (3×F-RNF8). WT EJ7-GFP and EJ6-GFP+TREX2 values are from Figure and shown here for comparison. As with all mESC experiments in this study, the reporters are integrated into chromosome 17 at the Pim1 locus. Error bars represent SD. N ≥ 6. (*) P ≤ 0.038 using unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot staining for Flag, and Actin control, in Rnf8-/- mESCs transfected with and without 3×F-RNF8. (*) Non-specific bands. ( D ) RNF8 promotes ALT-EJ in mESCs, as measured by the EJ2-GFP reporter. Shown are GFP+ frequencies for EJ2-GFP in WT and Rnf8-/- mESCs with and without complementation vector. N = 12. Error bars represent SD. (*) P ≤ 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( E ) RNF8 promotes ALT-EJ in HEK293 cells, as measured by the EJ2-GFP reporter. Two independent RNF8 knockout ( RNF8-KO ) cell lines ( RNF8-KO clones A and B) were generated in an HEK293 cell line with the EJ2-GFP reporter. Shown are GFP+ frequencies for the EJ2-GFP assay for these two cell lines with and without an RNF8 expression vector, as well as for the HEK293 WT cell line. N = 6. Error bars represent SD. (*) P ≤ 0.001766 for unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot analysis for RNF8, and Actin control, for the two RNF8-KO HEK293 cell lines transfected with RNF8, or only control EV, along with HEK293 WT cells transfected with EV.

Article Snippet: The pCAGGS-53BP1 (human) expression vector was generated from N-Myc-53BP1 WT pLPC-Puro (Addgene 19836) ( ).

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, MANN-WHITNEY, Transfection, Expressing, Plasmid Preparation, Western Blot, Knock-Out, Clone Assay

Journal: Nucleic Acids Research

Article Title: RNF8 has both KU-dependent and independent roles in chromosomal break repair

doi: 10.1093/nar/gkaa380

Figure Lengend Snippet: 53BP1 does not have a substantial effect on either EJ without indels or ALT-EJ, whereas POLQ promotes ALT-EJ independently of KU. ( A ) Influence of 53BP1 on distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, 53bp1-/- mESCs and 53bp1-/- mESCs transfected with an expression vector for human 53BP1. Also shown are GFP+ frequencies for 4-μHOM Embed in 53bp1-/-Rnf8-/- mESCs transfected with and without 53BP1 and RNF8 (3×F-RNF8) expression vectors, and compared to 53bp1-/- mESCs. WT values for EJ7-GFP and 4-μHOM are from Figures and , respectively, and are shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P ≤ 0.049 using unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot staining for 53BP1 and Actin control in WT and 53bp1-/- mESCs transfected with and without 53BP1. (*) represent non-specific bands. ( B ) Influence of POLQ on distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, Polq-/- mESCs and Polq-/- mESCs transfected with human Flag-tagged POLQ (POLQ). WT values for EJ7-GFP and 4-μHOM are from Figures and , respectively, and are shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P < 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( C ) Immunoblot analysis of KU70, Flag-POLQ and Actin control. A double mutant Ku70-/-Polq-/- mESC line was generated to examine the influence of POLQ and KU70 on EJ independently of the other factor. Shown are KU70 immunoblot signals for WT, and Ku70-/-Polq-/- mESCs transfected with and without KU70. Also shown are Flag and Actin control immunoblot signals for Ku70-/-Polq-/- mESCs transfected with and without Flag-tagged POLQ. (*) indicates a non-specific band. ( D ) POLQ and KU70 independently mediate distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, and Ku70-/-Polq-/- mESCs without complementation vector, or with expression vectors for KU70 or POLQ. WT values for EJ7-GFP, and 4-μHOM are from Figures and , respectively, and shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P < 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( E ) Shown is a model for the influence of RNF8 on chromosomal break repair outcomes.

Article Snippet: The pCAGGS-53BP1 (human) expression vector was generated from N-Myc-53BP1 WT pLPC-Puro (Addgene 19836) ( ).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Mutagenesis, Generated