Fig 1.
Alkbh8-/- MEFs have slow growth and increased apoptosis phenotypes.
A) 4 x 104 wt or Alkbh8 -/- MEFs were seeded per 9.6 cm2 well and viable (trypan blue negative) cells were counted over a period of 12 days. Error bars represent standard deviation (±STDV, n = 3) and significant differences in growth was determined by Student t-test (τp < 0.005, *p < 0.05). B) 1 x 104 cells were seeded per 58.1 cm2 dish, stained with crystal violet after 2 weeks in culture and colonies with a diameter > 2 mm were counted. Representative pictures of crystal violet stained cells. C-D) MEFs were allowed to grow to confluence and 2.5 x 105 cells were plated per 58.1 cm2 dish. Cells were fixed and assessed for apoptosis by TUNEL assay using a dUTP-FITC conjugate, 4 and 7 days after plating. Apoptotic cells were enumerated by flow cytometry and appear in the FITC-A channel. The percentage of TUNEL-positive cells is shown as the mean ± standard deviation (n = 3, right panel, *p < 0.05, **p < 0.001). E-F) RNA or whole protein extracts were prepared from sub-confluent cultures of wt and Alkbh8-/- MEFs and assessed for (E) PERP gene expression by qRT-PCR, in which each sample was normalized to internal Gapdh levels and (F) p53 protein expression by Western blotting
Fig 2.
Increased DNA damage and activated DNA damage response in Alkbh8-/- MEFs.
A) Comet assay of wt and Alkbh8-/- nuclei isolated from MEFs that were cultured as described above. B) Comet tails were quantified using CometScore and are shown as the mean % of DNA in the comet tail (±STDV, n = 3). Significant differences in the % of DNA in the comet tails of wt and Alkbh8-/- MEFs were determined by Student t-test *p < 0.001). C) Single cell suspensions were fixed and stained with a FITC conjugated antibody specific for γ-H2AX and the DRAQ5 nuclear stain followed by quantitative imaging with an ISX100 flow cytometer. Representative γ-H2AX foci images are shown for cell batches taken from the peak frequency of Spot Count distributions for wt and Alkbh8-/- sample runs. D) γ-H2AX foci were scored for intensity and number using the IDEAS Spot Count analysis program, presented as the percentage frequency of cells with 0–10 Spot Counts, and those with a Spot counts less than or greater than 3 (inset, n = 6).
Fig 3.
Alkbh8-/- MEFs are sensitive to DNA damaging agents.
MEFs were either treated with (A) MMS, (B) exposed to γ-irradiation or treated with (C) H2O2 or (D) Rotenone and viability was assessed by enumerating PI or trypan blue negative cells 48 (MMS, irradiation, and Rotenone) or 72 (H2O2) h later. In all panels, error bars represent standard deviation (±STDV, n = 3) and significant differences in viability of wt and Alkbh8-/- MEFs were determined by Student’s t-test (τp < 0.005, *p < 0.05, ψp < 0.01, ωp < 0.001).
Table 1.
Microarray-based transcript analysis was performed on early passage (P3) Alkbh8-/- and wt MEFs.
Fold change is the mean fluorescence intensity of Alkbh8-/- MEFs relative to wt MEFs (n = 3, p < 0.05).
Fig 4.
Alkbh8-/- cells have elevated reactive oxygen species (ROS).
A) Intracellular ROS was measured in immortalized MEFs by DCFDA staining. Representative images are shown for cell batches taken from the peak frequency of DCFDA intensities for wt and Alkbh8-/- MEFs. B) The frequency of wt (blue) and Alkbh8-/- (green) cells exhibiting specific DCFDA intensities is plotted for a population of 6,000 cells. C) Intracellular ROS was measured in immortalized MEFs by DCFDA staining 6 h after the indicated treatments and is shown as median fluorescence intensity emitted by oxidized DCFDA at 517–527 nm +STDV, n = 3).
Fig 5.
Compromised ROS response and stop-codon recoding with decreased Gpx and TrxR1 protein levels and activity in Alkbh8-/- MEFs.
Wt and Alkbh8-/- MEFs were treated as indicated and then prepared for western blot analysis with A) anti-Alkbh8, anti-Nrf2, anti-ATM S15 phosphorylated B) anti-Gpx, C) and anti-TrxR antibodies. Anti-TrXR1 antibody (Peirce PAS-28886) detects TrxR1 at 67 kD and a cross-reacting (higher MW) band in mouse cells. For A—C, anti-GAPDH antibodies were used to as loading controls. D) qRT-PCR analysis of Gpx1 transcript levels. E) qRT-PCR analysis of TrxR1 transcript levels. F) Lipid peroxidation levels were measured in whole cell lysates prepared from wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. G) Stop-codon recoding was measured using the DualLuc-Gpx1 reporter system in wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. Statistical significance (p < 0.05) of biological replicates (N = 3) was measured using the Student’s t-Test.
Table 2.
Settings of the Agilent 6430 triple quadrupole mass spectrometer for ribonucleoside analysis.
Fig 6.
Ribonucleoside modifications in wild type and Alkbh8-/- mice after oxidative stress.
Wt and Alkbh8-/- MEFs were either left untreated (UT) or exposed to 1.2 mM of H2O2 for 1 hour. The levels of mcm5s2U, mcm5Um and mcm5U ribonucleoside modifications were identified by HPLC-coupled mass spectrometry and quantified by integrating the normalized peak area intensity for each signal at the indicated post-exposure time points. Significant differences in mcm5Um modifications were determined by the Student’s t-test: UT, Alkbh8-/- (n = 6) versus wt (n = 5), *p < 0.05; 3 h, Alkbh8-/- (n = 3) versus wt (n = 3), **p < 0.02; 6 h, Alkbh8-/- (n = 3) versus wt (n = 3), ***p < 0.002; 20 h, Alkbh8-/- (n = 2) versus wt (n = 2), ****p < 0.01; 24 h, Alkbh8-/- (n = 3) versus wt (n = 2), *****p < 0.001. UT, 6 h, wt (n = 3) versus 0 hours, wt γp < 0.02; 20 hours, wt (n = 2) versus 0 h, wt (n = 5) γγp < 0.01.
Fig 7.
Model for Alkbh8 as a translational regulator of an ROS response node.
Alkbh8-directed cytoprotective responses through stop codon reprogramming for selenoprotein expression, and implications for disease prevention.