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. 2018 Jul 16;46(16):8326–8346. doi: 10.1093/nar/gky641

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — Ectopic expression of either SMN or SETX reduce R-loop accumulation and rescue DNA damage phenotype in SMA patient cells. Dividing SMA patient dividing cells, GM03813 and GM09677 (SMA) were transfected with pCMV/mycSMN or infected with Ad-h-SETX virus and stained with antibodies against SMN, phospho-H2AX (γH2AX), phospho-DNA-PKcs (p-DNAPKcs) and SETX. (A–D) Transient expression of SMN restores levels of SETX and p-DNA-PKcs and reduces R-loop and DNA damage accumulation in SMA patient cells. (A) SMN (green) and SETX (red), (B) SMN (green) and p-DNAPKcs (red), (C) R-loops (green) and p-DNAPKcs (red) and (D) SMN (green) and γH2AX (red). (E and F) Transient expression of SETX also reduces R-loops and DNA damage accumulation, and rescues phenotype of SMA patient cells. (E) SETX (red) and R-loops (green) and (F) SETX (red) and γH2AX (green). Nuclei were stained with DAPI (blue). Scale bar is 5.0 μm. Arrows show transfected cells and asterisks indicate non-transfected cells. (G) Effect of SMN overexpression on changes in the levels of SETX, γH2AX, p-DNAPKcs, total DNAPKcs in SMA patient cells GM03813 and GM09677 examined by IB analysis (full-length blots are included inSupplementary Figure S11). Quantitation and comparison of SMN, γH2AX, SETX, p-DNA-PKcs and DNA-PKcs levels in (H) GM03813 and GM03813+hSMN cells show rescue of DNA damage in SMN complemented cells (GM03813+hSMN) by marked reduction in γH2AX levels to (29.55 ± 4.22%, P = 0.000) compared to control GM03813 cells (120.5 ± 7.85%) that is supported by increase in levels of SETX (64.87 ± 15.37%, P = 0.013), p-DNA-PKcs (71.97 ± 20.83%, P = 0.025) and total DNA-PKcs (69.50 ± 18.91%, P = 0.021) and (I) analysis of GM09677 and GM09677+hSMN cells show rescue of DNA damage in SMN complemented cells (GM09677+hSMN) by marked reduction in γH2AX levels to (21.63 ± 5.76%, P = 0.002) compared to control GM09677 cells (124.3 ± 13.99%) that is supported by increase in levels of SETX (64.12 ± 15.97%, P = 0.015), p-DNA-PKcs (61.52 ± 7.62%, P = 0.001) and total DNA-PKcs (83.77 ± 18.93%, P = 0.011).