Fig 2: Alkbh7 deficiency confers partial resistance against MMS-induced toxicity in retina photoreceptors. (a) H&E stained representative sections of WT or Alkbh7-/- retina treated with either vehicle control or 75 mg/kg MMS and harvested at day 7. INL, inner nuclear layer. (b) Relative damage of the ONL assessed by histopathologic examination. Samples were categorized by increasing intensity of retinal degeneration ranging from minimal to severe and analyzed by chi-squared test. (c) Alkylation-induced retinal degeneration as determined by the thickness of the ONL. The number of cells in the ONL were counted for 25 representative areas of four sections of H&E-stained retina of the indicated mouse strains 7 days post-injection with 75 mg/kg MMS. Plotted is the average number of cells per ONL for each individual mouse, the mean (WT=4.3, Alkbh7-/-=5.9, and Aag-/-=11.2) and S.D. for each genotype. *P<0.05; **P<0.01; ***P<0.001

Fig 3: Sex-specific effects of ALKBH7 deficiency on whole-body MMS-toxicity. (a) Approximate LD50 of the indicated mouse strains in response to MMS. Four independent studies were performed. (b) Representative images (20X) of bone marrow samples extracted from femurs 24 h following MMS treatment (150 mg/kg). (c) Depletion of bone marrow is indistinguishable between WT and Alkbh7-/- mice following MMS-induced damage. The percent bone marrow depletion was determined by pathological examination of bone marrow samples as shown in (b). (d) Ex vivo bone marrow clonogenic survival assays were performed using bone marrow isolated from WT (n=6), Alkbh7+/- (n=6) and Alkbh7-/-(n=6) mice. Data represent the mean and S.D. For each of the genotypes, three male and three female mice were used in the experiments

Fig 4: Cellular sensitivity of isolated cerebellar neurons from WT or Alkbh7-/- mice in response to MMS. (a) Procedure for isolating cortical granule neurons (CGNs) from 6- to 8-day-old mouse pups followed by MMS treatment and viability analysis by microscopy. (b) Viability of the indicated CGNs after treatment with vehicle MMS or MMS with the caspase inhibitor, z-vad-fmk. (c) Viability of the indicated CGNs after treatment with vehicle MMS or MMS with the PARP1/ARTD1 inhibitor, veliparib. Data presented are from biological replicates, where each data point is the average of 2–4 wells with each well imaged 6–7 times

Fig 5: Alkbh7-/- mice display a sexually dimorphic response to alkylation-induced cerebellar degeneration. (a) H&E-stained cerebellar granule tissue showing the spectrum of responses in mice 24 h after mock treatment (control) or injection with 150 mg/kg MMS. The relative level of damage was scored by pathological classification of CGN cell death based upon the occurrence of pyknotic nuclei (yellow arrows). (b) Bar graph representing the relative percentage of cerebellar pyknosis of each mouse strain after alkylation exposure as scored in (a). (c) Quantification of cellular death in the cerebellar granular layer of the indicated male or female mouse strains 24 h post-treatment with 150 mg/kg MMS. The percent pyknotic nuclei represents the average of the number of pyknotic nuclei per total number of cells across 15 fields of H&E-stained brain sections of an individual mouse strain. The mean (males: WT=12.4, Alkbh7-/-=5.3; females: WT=8.6, Alkbh7-/-=12.2) and S.D. is represented for each genotype. *P<0.05; **P<0.01; ***P<0.001