Journal: ACS Central Science

Article Title: A Lamin-Binding Ligand Inhibits Homologous Recombination Repair of DNA Double-Strand Breaks

doi: 10.1021/acscentsci.8b00379

Figure Lengend Snippet: LBL1 inhibited HR. (A) LBL1 inhibited Rad51 subnuclear foci formation stimulated by CPT. MDA-MB-231 cells were treated as described at the top. Then the cells were analyzed by immunofluorescence analysis. Representative images are shown. (B) Quantification of Rad51-foci-positive cells from part A. Data are presented as mean ± SD ( n = 3, ∼100 cells were analyzed for each experimental condition). (C) LBL1 inhibited HR as assessed in a GFP-based reporter assay in MDA-MB-231 cells. The cells were transfected with a GFP-based HR reporter and DsRed as described in the . DSBs were induced by expressing I-SceI . Then the cells were treated with indicated concentrations of LBL1 . The cells were then analyzed by flow cytometry. The ratio of GFP + /DsRed + was registered as relative HR efficiency with vehicle-treated cells defined as 1.0 ( n = 3). (D, E) Overexpression of Rad51 rescued LBL1 ’s antiproliferative activity in MDA-MB-231 cells. FLAG-tagged Rad51 was overexpressed in MDA-MB-231 cells, and the cell lysates were analyzed by Western blot with indicated antibodies. (E) The cells were treated with indicated drugs for 48 h. The cellular growth was quantified by the MTT assay. Data are presented as mean ± SEM ( n = 3). * denotes P < 0.05.

Article Snippet: The plasmid expressing I-SceI (pCBASceI) and the plasmid expressing DsRed (DsRed2-N1) were from Addgene.

Techniques: Immunofluorescence, Reporter Assay, Transfection, Expressing, Flow Cytometry, Over Expression, Activity Assay, Western Blot, MTT Assay

Journal: bioRxiv

Article Title: Targeting Wnt Signaling To Overcome PARP Inhibitor Resistance

doi: 10.1101/378463

Figure Lengend Snippet: A ) PEO1 sensitive and PEO1-OR (resistant) cells were irradiated (5Gy) and incubated for the indicated time. Protein was extracted, separated with a SDS-PAGE and immunoblotted against of serine 139 phosphorylated histone H2x (γH2Ax). Loading control = β-actin. B ) Densitometric analysis of immunoblots. Relative γH2Ax levels calculated based on β-actin levels graphed over time analyzed with a linear regression. Slope is graphed with standard error. C ) Two-plasmid functional assay performed to assess distal non-homologous end joining. PEO1 sensitive and PEO1-OR clones were stably transfected with pimEJ5GFP and subsequently transfected with I-SceI restriction enzyme. After 72 hours transfected cells were collected and examined via a flow cytometer to quantify GFP positive cells. Representative gating strategy shown. D ) Same as C, Distal NHEJ GFP positive cells measured for PEO1 and PEO1-OR clones. P-values calculated with ANOVA. E ) Same as D, but utilized DRGRP to measure homology directed repair in PEO1 and PEO1-OR clones. P-values calculated with ANOVA. F ) Same as D, but cells treated with Pyr. Pam. 24hrs after I-SceI transfection. G ) Same as E, but treated with Pyr. Pam 24hrs after I-SceI transfection. Experiments performed in triplicate. Unless noted statistical used to calculate p-values, unpaired two tailed t-test. Error bars = SEM.

Article Snippet: After 72 hours post-I-SceI transfection, cells were collected and examined via a flow cytometer to quantify GFP positive cells. pimEJ5GFP and EJ2GFP were gifts from Jeremy Stark (Addgene plasmids # 44026 and # 44025). pDRGFP and pCBASceI were gifts from Maria Jasin (Addgene plasmid # 26475 and #26477).

Techniques: Irradiation, Incubation, SDS Page, Western Blot, Plasmid Preparation, Functional Assay, Non-Homologous End Joining, Clone Assay, Stable Transfection, Transfection, Flow Cytometry, Two Tailed Test