Fig. 1

Biological effects of SAMHD1 dysfunction in DNA damage response in CLL. (A) Plots depicting GO analysis results of the top significantly enriched pathways correlated with SAMHD1 expression in CLL patients. Arrows emphasize the biological processes related with DNA damage response. (B) Left panel: HR efficiency graph for DSB repair in PGA1 cell line using a HR reporter plasmid. HR repair efficiency was calculated as the ratio of GFP + cells to the total number of DsRed + (transfected) cells. Three clones per condition in 3 independent experiments were analyzed. Results are shown as mean ± SD. Right panel: Representative plots of HR repair in SAMHD1^WT and SAMHD1^KO cells. (C) Correlation analysis between BRCA1 and SAMHD1 mRNA expression normalized with the vst score in CLL patients from the CLL map project. (D) Left panel: Quantification of BRCA1 foci/cell in SAMHD1^WT and SAMHD1^KO cells 1-h post-IR. At least fifty cells were counted per experiment in two independent clones per condition. Right panel: Representative images of BRCA1-positive cells 1 h after irradiation (2 Gy). (E) Representative immunofluorescence images showing the colocalization of SAMHD1 with BRCA1 at DNA damage sites following 2 Gy irradiation. Images include untreated control (no irradiation) and irradiated (DNA-damage induced) conditions. (F) Left panel: Measurement of RAD51 foci/cell in γH2AX positive cells 6 h post-IR. Cells were considered as γH2AX + when 5 or more foci were formed. At least fifty cells were counted per experiment. Two independent experiments were performed in three biological replicates. Right panel: Representative images of γH2AX + cells and RAD51 localization. (G) Western blot analyses depicting the expression of the proteins implicated in DNA damage signaling ATM, ATR, CHK1 and CHK2; and their phosphorylated forms. Cells were not treated (0), or treated with 2 or 4 µM of etoposide for 8 h. Two single-cell clones were analyzed for each condition.