Figure 4: Survival, replication of bulky DNA lesions and DNA damage signaling in Rev3l-mutated MEF lines.
From: De novo mutations in PLXND1 and REV3L cause Möbius syndrome
(a) Survival of wt (+/+), Rev3l heterozygous (+/−) and Rev3l-deficient (−/−) MEF lines after mock exposure, or after exposure to Benzo(a)pyrene diolepoxyde (BPDE). Rev3l heterozygous MEFs are slightly sensitive to BPDE indicating haploinsufficiency. Experiments were performed in triplicate. Error bars, s.e.m. The asterisks indicate a significant difference between the wt and the heterozygous lines (unpaired t-test, P<0.05). (b) Representation of replication fork progression after exposure to BPDE, or mock exposure. Rev3l heterozygous MEFs display normal fork progression at undamaged DNA (mock treated, left panel), but a marginal defect in replication of bulky lesion-containing DNA (BPDE, right panel). Error bars, s.e.m. (c) Immunoblots to detect the phosphorylation of DNA damage-signalling markers upon treatment with BPDE. γ-H2AX: phosphorylated histone H2AX. RpaS4/8-P: 32 kDa subunit of single-stranded DNA-binding protein Rpa, phosphorylated at Ser 4 and/or Ser 8. Total levels of Rpa are somewhat variable consequent to slight differences in proliferation rate between the lines. Chk1S354-P Checkpoint kinase-1, phosphorylated at Ser 354. β-actin: loading control. Rev3l-heterozygous MEFs display DNA damage signalling that is almost as strong as Rev3l-deficient MEFs.
