Research Paper Volume 11, Issue 23 pp 11268—11313

Figure 9. RXFP3 acts as a protective factor of DNA damage. (A) RXFP3 expression increases significantly with DNA damage caused by 10 μM CPT for 24 hours (n=3). Induction of DNA damage was validated using γ-H2AX and p-ATM. (B) Overexpression of RXFP3 compared to control (E.V.) elicits a specific response of DNA damage-associated proteins in unstressed stressed (CPT-treated) cells. Overexpression of the RXFP3, in the absence of CPT exposure, appears to prepare the cell for stress responsivity as this results in the activation of BRCA1 (p-BRCA1), PRKDC (p-PRKDC), ATM (p-ATM) and H2AX (γ-H2AX). However, we also see a decrease in activation compared to control (E.V.) transfected cells after stress of these proteins, indicating that the RXFP3 potentially facilitates DNA damage repair (n=3). (C) RXFP3 stimulation using RLN3 (100 nM, 1 and 2 hours prior to stress induction using 10 μM CPT), directly affects the number of γ-H2AX, pBRCA1 and pPRKDC foci (n=30) using confocal microscopy, where we see a specific decrease in γ-H2AX, and p-BRCA1 foci, and an increase in PRKDC activation. (D) These results were also shown using immunoblotting (n=6). (E) Stimulating RXFP3 after stress induction (2 shots, 1 and 2 hours after 3h 10 μM CPT), here called post-stimulation, induced similar effects (n=6).