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. 2020 Sep 14;106(11):2906–2917. doi: 10.3324/haematol.2020.254193

Figure 2.

Figure 2.

Elevated levels of R-loops induce replication stress in splicing factor mutated myelodysplastic syndrome CD34+ cells. (A) Experimental procedure and representative images of CldU/IdU labeled DNA fibers. (B) Quantification of DNA fork velocity in CD34+ cells isolated from myelodysplastic syndrome (MDS) patient bone marrow with and without splicing factor (sf) mutations as well as healthy controls (young and old). n=441 (non-sf mutated), n=400 (sf mutated), n=279 (young CD34+), n=290 (old CD34+). (C) Schematic illustration of the DNA damage response pathway activated in response to replication stress and representative immunofluorescence images of pRPA (S33) in sf mutated and non-sf mutated MDS CD34+ cells. Scale bar, 5 μm. (D) Schematic illustration of the DNA damage response pathway activated in response to replication stress. (E) Representative immunofluorescence images of pRPA (S33) in healthy young, healthy old, MDS sf mutated, and MDS non-sf mutated CD34+ cells. Scale bar, 5 μm. (F) Quantification of pRPA (S33) mean fluorescence intensity (MFI). n=7 (373 cells, non-sf mutated), n= 13 (522 cells, sf mutated), n=4 (279 cells, young CD34+), n=6 (290 cells, old CD34+). Data are means +/- standard deviation. *P≤ 0.05; *** P≤0.001; n.s.: not significant; CldU: 5-chloro-2′-deoxyuridine; IdU: 5-iodo-2′-deoxyuridine (IdU); A.U: arbitrary units.