brca2  (Proteintech)

Journal: PLOS One

Article Title: Differential sensitivity of MCPH1- and BRCA2-deficient cancer cells to PARP-1 inhibition

doi: 10.1371/journal.pone.0345514

Figure Lengend Snippet: (A) . U2OS or Hela cells were transfected with Control (siCON), MCPH1 (siMCPH1) or BRCA2 (siBRCA2) siRNA for 48h or 120h and protein expression analysed via Western blotting with the indicated antibodies. MCPH1 expression in sgCON (control) and sgMCPH1 (MCPH1 knockout) cells was also investigated by Western blotting. (B) Quantification of MCPH1 or BRCA2 protein bands normalised to GAPDH and expressed as % change relative to siCON or sgCON. Data is the mean and standard error of three (48h) or two (120h and sgCON/sgMCPH1) independent experiments. (C) U2OS or Hela cells were transfected with MCPH1 siRNA for 72h before analysis of nuclear morphology (premature chromosome condensation, PCC) via DAPI staining and fluorescence microscopy. (D) The % of cells displaying PCC, with at least 100 cells analysed.

Article Snippet: Membranes were blocked with 5% dried skimmed milk/TBS-T (50mM Tris pH 7.6, 150mM NaCl and 0.2% v/v Tween-20) for a minimum of 1-hour prior to overnight incubation at 4°C with primary antibodies; MCPH1 (11962–1-AP, Proteintech), BRCA2 (29450–1-AP, Proteintech) and GAPDH (60004–1-Ig, Proteintech).

Techniques: Transfection, Control, Expressing, Western Blot, Knock-Out, Staining, Fluorescence, Microscopy

Journal: PLOS One

Article Title: Differential sensitivity of MCPH1- and BRCA2-deficient cancer cells to PARP-1 inhibition

doi: 10.1371/journal.pone.0345514

Figure Lengend Snippet: U2OS cells were transfected with control (siCON), BRCA2 (siBRCA2) or BRCA2 + MCPH1 (siBRCA2 + siMCPH1) siRNA for 48h before (A) Western blot analysis or (B) seeding into 96-well plates and treating with the indicated doses of AZD2461 or Talazoparib, with each treatment performed in triplicate. After 96-hours, cell viability was measured using an MTS assay. MTS data represents the mean and standard error from at least three independent experiments, with cell viability expressed as a % relative to the corresponding untreated sample. Statistical significance was measured using two-way ANOVA (p-values described in text, with significance set at p < 0.05).

Article Snippet: Membranes were blocked with 5% dried skimmed milk/TBS-T (50mM Tris pH 7.6, 150mM NaCl and 0.2% v/v Tween-20) for a minimum of 1-hour prior to overnight incubation at 4°C with primary antibodies; MCPH1 (11962–1-AP, Proteintech), BRCA2 (29450–1-AP, Proteintech) and GAPDH (60004–1-Ig, Proteintech).

Techniques: Transfection, Control, Western Blot, MTS Assay

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARPi-dependent MMEJ increase depends on HR. ( A ) MMEJ quantification using the reporter and experimental timeline from Fig. and in HT1080 cells following treatment with olaparib (5 µM), DNA-PKcsi (NU7441, 1 µM), or both (combo). Values are normalized to DMSO. Immunoblot of 53BP1 and histone H3 in parental HT1080 cells and isogenic TP53BP1 -KO cells. ( C ) MMEJ quantification in HT1080 parental cells and indicated isogenic 53BP1 -KO cells. Values are normalized to wild-type DMSO. Drug used: olaparib (5 µM). ( D ) Immunoblot of BRCA2 and actin in parental HT1080 cells and two isogenic BRCA2 -KO clonal lines. ( E ) MMEJ quantification in HT1080 parental cells and BRCA2 -KO clones. Values are normalized to wild-type DMSO. Drug used: olaparib (5 µM). Statistical analyses for panels (A), (C), and (E). Data represent three independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, * P <.05, ** P <.01, **** P <.0001.

Article Snippet: Antibodies used for immunoblotting: PARP1 (diluted 1:2000, Cell Signaling Technology #9542), PARP2 (diluted 1:2000, Active Motif #39744, Antibody has been discontinued), β-Actin (diluted 1:5000, Cell Signaling Technology #8457), WRN (diluted 1:2000, Cell Signaling Technology #4666, RRID:AB_10692114 ), EXO1 (diluted 1:1000, Cell Signaling Technology #63862), PAR (diluted 1:2000, Cell Signaling Technology #87733), 53BP1 (diluted 1:2000, Novus Biologicals #NB100-304), H3 (diluted 1:5000, Cell Signaling Technology #9715), anti-mouse IgG, HRP-linked (diluted 1:5000, Cell Signaling #7076), anti-rabbit IgG, HRP-linked (diluted 1:5000, Cell Signaling #7074), BRCA2 (diluted 1:1000, Bethyl #A303-434A, RRID:AB_10952240 ).

Techniques: Western Blot, Clone Assay, Comparison