Fig. 8: Decreased BRCA2 levels in Atg12-deleted cells result in increased DNA damage and centrosome abnormalities.

a Immunofluorescence for γH2AX (red) in Atg12^f/f and Atg12^KO MEFs; nuclei are counterstained with Hoechst (blue). Bar = 50 μm. Percent of γH2AX-positive cells was quantified in Atg12^f/f and Atg12^KO MEFs; three random fields were counted over three independent biological replicates, p = 0.002 by t test. b Immunofluorescence for γH2AX (red) and 53BP1 (green) in Atg12^f/f and Atg12^KO MEF; nuclei are counterstained with Hoechst (blue). Bar = 50 μm. Three biologically independent replicates were performed. c ROS-glo assay (Promega) in Atg12^f/f and Atg12^KO MEFs treated with vehicle control or menadione (50 μM for 2 h, positive control) (mean ± SEM, n = 2 biologically independent replicates). d Protein lysate was collected from Atg12^f/f and Atg12^KO MEFs treated with vehicle control or NAC (5 mM for 8 h), or ectopically overexpressing either GFP (pGFP) or human BRCA2 (huBRCA2). A representative immunoblot for γH2AX is shown, as well as boxplots with independent biological replicates, for γH2AX levels normalized to loading control. Statistical analysis was performed by t test. e Protein lysate was collected from Atg12^f/f and Atg12^KO MEFs treated for 16 h with vehicle control, rucaparib (100 nM), olaparib (100 nM), or BMN (2 nM), and immunoblotted as shown. Three independent biological replicates were performed. f A clonogenic replating assay was performed on Atg12^f/f or Atg12^KO MEFs treated for 16 h with vehicle control, rucaparib (100 nM), olaparib (100 nM), or BMN (2 nM). Colony number is shown as a boxplot including biological replicates, p value by t test, g Immunofluorescence of centrosomes (γ-tubulin, red) and mitotic cells (pH3, green), nuclei counterstained by Hoechst (blue), in Atg12^f/f and Atg12^KO MEFs. Yellow box indicates magnified region below. White arrows indicate non-mitotic cells with multiple centrosomes (3+) or non-clustered centrosomes. Bar = 100 μm. h Quantification (mean ± SEM, n = 3 biologically independent replicates) of distance between mother and daughter centrosomes measured on immunofluorescence of γ-tubulin in non-pH3 positive cells, p value by t test. i Quantification of Atg12^f/f and Atg12^KO MEFs with abnormal numbers (3+) of centrosomes from immunofluorescence images (n = 4 biologically independent replicates). p values by t test on logit transformed percent per replicate. Fraction above the bar plots indicates number of cells with 3+ centrosomes out of number of cells enumerated.